Bolaamphiphilic compounds, compositions and uses thereof

ABSTRACT

Bolaamphiphilic compounds are provided according to formula I: 
       HG 2 -L 1 -HG 1    I
 
     where HG 1 , HG 2  and L 1  are as defined herein. Provided bolaamphilphilic compounds and the pharmaceutical compositions thereof are useful for delivering biologically active drugs into animal or human brain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/638,466 filed Mar. 4, 2015, which is a continuation of InternationalApplication No. PCT/US2013/057960 filed Sep. 4, 2013, which claimspriority to U.S. Application No. 61/696,798, filed Sep. 4, 2012, thecontents of which are incorporated by reference herein. U.S. applicationSer. No. 14/638,466, filed Mar. 4, 2015, also claims the benefit of U.S.Application No. 61/974,201 filed Apr. 2, 2014, the contents of which arealso incorporated by reference herein.

FIELD

Provided herein are nanovesicles comprising bolaamphiphilic compounds,and complexes thereof with biologically active molecules, andpharmaceutical compositions thereof. Also provided are methods ofdelivering biologically active molecules into the human brain and animalbrain using the compounds, complexes and pharmaceutical compositionsprovided herein.

BACKGROUND

Many drugs and biologically active molecules cannot penetrate the BBBand thus require direct administration into the CNS tissue or thecerebral spinal fluid (CSF) in order to achieve a biological ortherapeutic effect. Even direct administration into a particular CNSsite is often limited due to poor diffusion of the active agent becauseof local absorption/adsorption into the CNS matrix. Present modalitiesfor drug delivery through the BBB entail disruption of the BBB by, forexample, osmotic means (hyperosmotic solutions) or biochemical means(e.g., use of vasoactive substances such as. bradykinin), processes withserious side effects.

The brain is a highly specialized organ, and its sensitive componentsand functioning are protected by a barrier known as the blood-brainbarrier (BBB). The brain capillary endothelial cells (BCECs) that formthe BBB play important role in brain physiology by maintaining selectivepermeability and preventing passage of various compounds from the bloodinto the brain. One consequence of the highly effective barrierproperties of the BBB is the limited penetration of therapeutic agentsinto the brain, which makes treatment of many brain diseases extremelychallenging.

Efforts to improve the permeation of biologically active compoundsacross the BBB using amphiphilic vesicles have been attempted.

For example, complexation of the anionic carboxyfluorescein (CF) withsingle headed amphiphiles of opposite charge in cationic vesicles,formed by mixing single-tailed cationic and anionic surfactants has beenreported (Danoff et al. 2007).

Furthermore, WO 02/055011 and WO 03/047499, both of the same applicant,disclose amphiphilic derivatives composed of at least one fatty acidchain derived from natural vegetable oils such as vernonia oil,lesquerella oil and castor oil, in which functional groups such asepoxy, hydroxy and double bonds were modified into polar and ionicheadgroups.

Additionally, WO 10/128504 reports a series of amphiphiles andbolamphiphiles (amphiphiles with two head groups) useful for targeteddrug delivery of insulin, insulin analogs, TNF, GDNF, DNA, RNA(including siRNA), enkephalin class of analgesics, and others.

These synthetic bolaamphiphiles (bolas) have recently been shown to formnanovesicles that interact with and encapsulate a variety of small andlarge molecules including peptides, proteins and plasmid DNAs deliveringthem across biological membranes. These bolaamphiphiles are a uniqueclass of compounds that have two hydrophilic headgroups placed at eachends of a hydrophobic domain. Bolaamphiphiles can form vesicles thatconsist of monolayer membrane that surrounds an aqueous core. Vesiclesmade from natural bolaamphiphiles, such as those extracted fromarchaebacteria (archaesomes), are very stable and, therefore, might beemployed for targeted drug delivery. However, bolaamphiphiles fromarchaebacteria are heterogeneous and cannot be easily extracted orchemically synthesized.

Thus, there remains a need to make new compositions and for novelmethods to deliver biologically active drugs into the brain. Thecompounds, compositions, and methods described herein are directedtoward this end.

SUMMARY OF THE INVENTION

In certain aspects, provided herein are pharmaceutical compositionscomprising of a bolaamphiphile complex.

In further aspects, provided herein are novel nano-sized vesiclescomprising of bolaamphiphilic compounds.

In certain aspects, provided herein are novel bolaamphiphile complexescomprising one or more bolaamphiphilic compounds and a biologicallyactive compound.

In one embodiment, the biologically active compound is a compound activeagainst ALS. In another embodiment, the biologically active compound isan analgesic compound.

In further aspects, provided herein are novel formulations ofbiologically active compounds with one or more bolaamphiphilic compoundsor with bolaamhphile vesicles.

In another aspect, provided here are methods of delivering biologicallyactive drugs agents into animal or human brain. In one embodiment, themethod comprises the step of administering to the animal or human apharmaceutical composition comprising of a bolaamphiphile complex; andwherein the bolaamphiphile complex comprises one or more bolaamphiphiliccompounds and a compound active against ALS. In one particularembodiment, the biologically active compound is an analgesic compound.

In one embodiment, the bolaamphiphilic compound consists of twohydrophilic headgroups linked through a long hydrophobic chain. Inanother embodiment, the hydrophilic headgroup is an amino containinggroup. In a specific embodiment, the hydrophilic headgroup is a tertiaryor quaternary amino containing group.

In one particular embodiment, the bolaamphiphilic compound is a compoundaccording to formula I:

HG²-L¹-HG¹   I

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

each HG¹ and HG² is independently a hydrophilic head group; and

L¹ is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker;unsubstituted or substituted with C₁-C₂₀ alkyl, hydroxyl, or oxo.

In one embodiment, the pharmaceutically acceptable salt is a quaternaryammonium salt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, the bolaamphiphilic compound is a compound according toformula II, III, IV, V, or VI:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

each HG¹ and HG² is independently a hydrophilic head group;

each Z¹ and Z² is independently —C(R³)₂—, —N(R³)— or —O—;

each R^(1a), R^(1b), R³, and R⁴ is independently H or C₁-C₈ alkyl;

each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH, alkoxy, orO-HG¹ or O-HG²;

each n8, n9, n11, and n12 is independently an integer from 1-20;

n10 is an integer from 2-20; and

each dotted bond is independently a single or a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, or VI, each HG¹ and HG² is independentlyselected from:

wherein:

-   -   X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a), and        R^(5b) is independently H or substituted or unsubstituted C₁-C₂₀        alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl; each R^(5c) is        independently substituted or unsubstituted C₁-C₂₀ alkyl; each R⁸        is independently H, substituted or unsubstituted C₁-C₂₀ alkyl,        alkoxy, or carboxy;    -   m1 is 0 or 1; and    -   each n13, n14, and n15 is independently an integer from 1-20.

In another embodiment, the present disclosure provides bolaamphiphiles,methods for the synthesis and use thereof, and compositions comprisingsame, that may be prepared from jojoba oil.

In another embodiment, the present disclosure provides bolaamphiphilesdescribed within this application, methods for the synthesis and usethereof, and compositions comprising same, that include cyclodextrinswithin the compositions that form vesicles.

In another embodiment, the present disclosure provides bolaamphiphilescomprising specific targeting ligands, methods for the synthesis and usethereof, and compositions comprising same, that may used, e.g., for thetreatment of brain tumors. In one aspect of this embodiment, thetargeted brain tumor is a glioblastoma multiforme (GBM).

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing detailed description.

FIGURES

FIG. 1: TEM micrograph of vesicles from GLH-20 (A) and their sizedistribution determined by DLS (B).

FIG. 2: Head group hydrolysis by AChE (A) of GLH-19 (blue) and GLH-20(red) and release of CF from GLH-19 vesicles (B) and GLH-20 vesicles (C)

FIG. 3: CF accumulation in brain after i.v. injection of encapsulatedand non-encapsulated CF. Only GLH-20 vesicles allow accumulation of CFin the brain (A). CS improves GLH-20 vesicles' penetration into thebrain (B).

FIG. 4: Analgesia after i.v. injection of enkephalin non-encapsulatedand encapsulated in vesicles. Analgesia (compared with morphine, whichwas used as a positive control) is obtained only when enkephalin isencapsulated in GLH-20 vesicles (A), the head groups of which arehydrolyzed by ChE. The vesicles do not disrupt the BBB sincenon-encapsulated enkephalin co-injected with empty vesicles(extravesicular enkephalin) did not cause analgesia (B). **Significantlydifferent from free leu-enkephalin (t-test, P<0.01). ***Significantlydifferent from free leu-enkephalin (t-test, P<0.001).

FIG. 5: Fluorescence in mouse cerebral cortex after i.v. injection ofalbumin-FITC (non-encapsulated) (A) encapsulated in GLH-20 vesicles (B).

FIG. 6: Brain delivery of analgesic peptide kyotorphin.

FIG. 7: ¹H-NMR and ¹³C-NMR of Compound Compound (4)

FIG. 8: (A) MALDI spectrum of jojoba dichloroacetate; (B) Comparisonbetween theoretical and actual disruption abundance of isotopes inC₄₆H₈₆Cl₂O₆.

FIG. 9: ¹H-NMR and ¹³C-NMR of the bolaamphiphile GLH-58.

FIG. 10: MS (ESI) ([M-2Cl]⁺/2) of bolaamphiphile GLH-58.

FIG. 11: ¹H-NMR and ¹³C-NMR spectrua of the tetrachloroacetate of jojobaoil (10).

FIG. 12: MALDI spectrum of tetracholoracetate of (A) jojoba oil(compound (10)) and (B) of the bolaamphiphile GLH-60.

DEFINITIONS Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in ThomasSorrell, Organic Chemistry, University Science Books, Sausalito, 1999;Smith and March, March's Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistryof Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen, Tables ofResolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ.of Notre Dame Press, Notre Dame, Ind. 1972). The invention additionallyencompasses compounds described herein as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention. When describing the invention,which may include compounds, pharmaceutical compositions containing suchcompounds and methods of using such compounds and compositions, thefollowing terms, if present, have the following meanings unlessotherwise indicated. It should also be understood that when describedherein any of the moieties defined forth below may be substituted with avariety of substituents, and that the respective definitions areintended to include such substituted moieties within their scope as setout below. Unless otherwise stated, the term “substituted” is to bedefined as set out below. It should be further understood that the terms“groups” and “radicals” can be considered interchangeable when usedherein. The articles “a” and “an” may be used herein to refer to one orto more than one (i.e. at least one) of the grammatical objects of thearticle. By way of example “an analogue” means one analogue or more thanone analogue.

“Alkyl” refers to a radical of a straight-chain or branched saturatedhydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀ alkyl”). Insome embodiments, an alkyl group has 1 to 12 carbon atoms (“C₁₋₁₂alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms(“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1 to 9 carbonatoms (“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1to 7 carbon atoms (“C₁₇ alkyl”). In some embodiments, an alkyl group has1 to 6 carbon atoms (“C₁₋₆ alkyl”, also referred to herein as “loweralkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms(“C₁₋₅ alkyl”). In some embodiments, an alkyl group has 1 to 4 carbonatoms (“C₁₋₄ alkyl”). In some embodiments, an alkyl group has 1 to 3carbon atoms (“C₁₋₃ alkyl”). In some embodiments, an alkyl group has 1to 2 carbon atoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl grouphas 1 carbon atom (“C₁ alkyl”). In some embodiments, an alkyl group has2 to 6 carbon atoms (“C₂₋₆ alkyl”). Examples of C₁₋₆ alkyl groupsinclude methyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl(C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅),3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅),tertiary amyl (C₅), and n-hexyl (C₆). Additional examples of alkylgroups include n-heptyl (C₇), n-octyl (C₈) and the like. Unlessotherwise specified, each instance of an alkyl group is independentlyoptionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents;e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1substituent. In certain embodiments, the alkyl group is unsubstitutedC₁₋₁₀ alkyl (e.g., —CH₃). In certain embodiments, the alkyl group issubstituted C₁₋₁₀ alkyl.

“Alkylene” refers to a substituted or unsubstituted alkyl group, asdefined above, wherein two hydrogens are removed to provide a divalentradical. Exemplary divalent alkylene groups include, but are not limitedto, methylene (—CH₂—), ethylene (—CH₂CH₂—), the propylene isomers (e.g.,—CH₂CH₂CH₂— and —CH(CH₃)CH₂—) and the like.

“Alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon double bonds, and no triple bonds (“C₂₋₂₀ alkenyl”). Insome embodiments, an alkenyl group has 2 to 10 carbon atoms (“C₂₋₁₀alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C₂₋₃alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C₂alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl(C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like.Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄ alkenylgroups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and thelike. Additional examples of alkenyl include heptenyl (C₇), octenyl(C₈), octatrienyl (C₈), and the like. Unless otherwise specified, eachinstance of an alkenyl group is independently optionally substituted,i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a“substituted alkenyl”) with one or more substituents e.g., for instancefrom 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Incertain embodiments, the alkenyl group is unsubstituted C₂₋₁₀ alkenyl.In certain embodiments, the alkenyl group is substituted C₂₋₁₀ alkenyl.

“Alkenylene” refers a substituted or unsubstituted alkenyl group, asdefined above, wherein two hydrogens are removed to provide a divalentradical. Exemplary divalent alkenylene groups include, but are notlimited to, ethenylene (—CH═CH—), propenylenes (e.g., —CH═CHCH₂— and—C(CH₃)═CH— and —CH═C(CH₃)—) and the like.

“Alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 20 carbon atoms, one or morecarbon-carbon triple bonds, and optionally one or more double bonds(“C₂₋₂₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 10carbon atoms (“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl grouphas 2 to 9 carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, analkynyl group has 2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In someembodiments, an alkynyl group has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”).In some embodiments, an alkynyl group has 2 to 6 carbon atoms (“C₂₋₆alkynyl”). In some embodiments, an alkynyl group has 2 to 5 carbon atoms(“C₂₋₅ alkynyl”). In some embodiments, an alkynyl group has 2 to 4carbon atoms (“C₂₋₄ alkynyl”). In some embodiments, an alkynyl group has2 to 3 carbon atoms (“C₂₋₃ alkynyl”). In some embodiments, an alkynylgroup has 2 carbon atoms (“C₂ alkynyl”). The one or more carbon-carbontriple bonds can be internal (such as in 2-butynyl) or terminal (such asin 1-butynyl). Examples of C₂₋₄ alkynyl groups include, withoutlimitation, ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl(C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆ alkenyl groupsinclude the aforementioned C₂₋₄ alkynyl groups as well as pentynyl (C₅),hexynyl (C₆), and the like. Additional examples of alkynyl includeheptynyl (C₇), octynyl (C₈), and the like. Unless otherwise specified,each instance of an alkynyl group is independently optionallysubstituted, i.e., unsubstituted (an “unsubstituted alkynyl”) orsubstituted (a “substituted alkynyl”) with one or more substituents;e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1substituent. In certain embodiments, the alkynyl group is unsubstitutedC₂₋₁₀ alkynyl. In certain embodiments, the alkynyl group is substitutedC₂₋₁₀ alkynyl.

“Alkynylene” refers a substituted or unsubstituted alkynyl group, asdefined above, wherein two hydrogens are removed to provide a divalentradical. Exemplary divalent alkynylene groups include, but are notlimited to, ethynylene, propynylene, and the like.

“Aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclicor tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 πelectrons shared in a cyclic array) having 6-14 ring carbon atoms andzero heteroatoms provided in the aromatic ring system (“C₆₋₁₄ aryl”). Insome embodiments, an aryl group has six ring carbon atoms (“C₆ aryl”;e.g., phenyl). In some embodiments, an aryl group has ten ring carbonatoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). Insome embodiments, an aryl group has fourteen ring carbon atoms (“C₁₋₄aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein thearyl ring, as defined above, is fused with one or more carbocyclyl orheterocyclyl groups wherein the radical or point of attachment is on thearyl ring, and in such instances, the number of carbon atoms continue todesignate the number of carbon atoms in the aryl ring system. Typicalaryl groups include, but are not limited to, groups derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, coronene, fluoranthene, fluorene, hexacene,hexaphene, hexalene, as-indacene, s-indacene, indane, indene,naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene,pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene,picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, andtrinaphthalene. Particularly aryl groups include phenyl, naphthyl,indenyl, and tetrahydronaphthyl. Unless otherwise specified, eachinstance of an aryl group is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted aryl”) or substituted (a “substitutedaryl”) with one or more substituents. In certain embodiments, the arylgroup is unsubstituted C₆₋₁₄ aryl. In certain embodiments, the arylgroup is substituted C₆₋₁₄ aryl.

In certain embodiments, an aryl group substituted with one or more ofgroups selected from halo, C₁-C₈ alkyl, C₁-C₈ haloalkyl, cyano, hydroxy,C₁-C₈ alkoxy, and amino.

Examples of representative substituted aryls include the following

In these formulae one of R⁵⁶ and R⁵⁷ may be hydrogen and at least one ofR⁵⁶ and R⁵⁷ is each independently selected from C₁-C₈ alkyl, C₁-C₈haloalkyl, 4-10 membered heterocyclyl, alkanoyl, C₁-C₈ alkoxy,heteroaryloxy, alkylamino, arylamino, heteroarylamino, NR⁵⁸COR⁵⁹,NR⁵⁸SOR⁵⁹NR⁵⁸SO₂R⁵⁹, COOalkyl, COOaryl, CONR⁵⁸R⁵⁹, CONR⁵⁸OR⁵⁹, NR⁵⁸R⁵⁹,SO₂NR⁵⁸R⁵⁹, S-alkyl, SOalkyl, SO₂alkyl, Saryl, SOaryl, SO₂aryl; or R⁵⁶and R⁵⁷ may be joined to form a cyclic ring (saturated or unsaturated)from 5 to 8 atoms, optionally containing one or more heteroatomsselected from the group N, O, or S. R⁶⁰ and R⁶¹ are independentlyhydrogen, C₁-C₈ alkyl, C₁-C₄ haloalkyl, C₃-C₁₀ cycloalkyl, 4-10 memberedheterocyclyl, C₆-C₁₀ aryl, substituted C₆-C₁₀ aryl, 5-10 memberedheteroaryl, or substituted 5-10 membered heteroaryl.

“Fused aryl” refers to an aryl having two of its ring carbon in commonwith a second aryl ring or with an aliphatic ring.

“Aralkyl” is a subset of alkyl and aryl, as defined herein, and refersto an optionally substituted alkyl group substituted by an optionallysubstituted aryl group.

“Heteroaryl” refers to a radical of a 5-10 membered monocyclic orbicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen and sulfur(“5-10 membered heteroaryl”). In heteroaryl groups that contain one ormore nitrogen atoms, the point of attachment can be a carbon or nitrogenatom, as valency permits. Heteroaryl bicyclic ring systems can includeone or more heteroatoms in one or both rings. “Heteroaryl” includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more carbocyclyl or heterocyclyl groups wherein the point ofattachment is on the heteroaryl ring, and in such instances, the numberof ring members continue to designate the number of ring members in theheteroaryl ring system. “Heteroaryl” also includes ring systems whereinthe heteroaryl ring, as defined above, is fused with one or more arylgroups wherein the point of attachment is either on the aryl orheteroaryl ring, and in such instances, the number of ring membersdesignates the number of ring members in the fused (aryl/heteroaryl)ring system. Bicyclic heteroaryl groups wherein one ring does notcontain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and thelike) the point of attachment can be on either ring, i.e., either thering bearing a heteroatom (e.g., 2-indolyl) or the ring that does notcontain a heteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently optionally substituted, i.e., unsubstituted (an“unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”)with one or more substituents. In certain embodiments, the heteroarylgroup is unsubstituted 5-14 membered heteroaryl. In certain embodiments,the heteroaryl group is substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatominclude, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing two heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing threeheteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing fourheteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing one heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containingtwo heteroatoms include, without limitation, pyridazinyl, pyrimidinyl,and pyrazinyl. Exemplary 6-membered heteroaryl groups containing threeor four heteroatoms include, without limitation, triazinyl andtetrazinyl, respectively. Exemplary 7-membered heteroaryl groupscontaining one heteroatom include, without limitation, azepinyl,oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groupsinclude, without limitation, indolyl, isoindolyl, indazolyl,benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl,indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude, without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

Examples of representative heteroaryls include the following:

wherein each Y is selected from carbonyl, N, NR⁶⁵, O, and S; and R⁶⁵ isindependently hydrogen, C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl, 4-10 memberedheterocyclyl, C₆-C₁₀ aryl, and 5-10 membered heteroaryl.

Examples of representative aryl having hetero atoms containingsubstitution include the following:

wherein each W is selected from C(R⁶⁶)₂, NR⁶⁶, O, and S; and each Y isselected from carbonyl, NR⁶⁶, O and S; and R⁶⁶ is independentlyhydrogen, C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl,C₆-C₁₀ aryl, and 5-10 membered heteroaryl.

“Heteroaralkyl” is a subset of alkyl and heteroaryl, as defined herein,and refers to an optionally substituted alkyl group substituted by anoptionally substituted heteroaryl group.

“Carbocyclyl” or “carbocyclic” refers to a radical of a non-aromaticcyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C₃₋₁₀carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. Insome embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms(“C₃₋₈ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 6 ring carbon atoms (“C₃₋₆ carbocyclyl”). Insome embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms(“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groups include,without limitation, cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl(C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like.Exemplary C₃ s carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or contain a fused, bridged orspiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) andcan be saturated or can be partially unsaturated. “Carbocyclyl” alsoincludes ring systems wherein the carbocyclyl ring, as defined above, isfused with one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclyl ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. Unless otherwise specified, each instance of acarbocyclyl group is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is unsubstituted C₃₋₁₀ carbocyclyl.In certain embodiments, the carbocyclyl group is a substituted C₃₋₁₀carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is unsubstituted C₃₋₁₀ cycloalkyl. Incertain embodiments, the cycloalkyl group is substituted C₃₋₁₀cycloalkyl.

“Heterocyclyl” or “heterocyclic” refers to a radical of a 3- to10-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 memberedheterocyclyl”). In heterocyclyl groups that contain one or more nitrogenatoms, the point of attachment can be a carbon or nitrogen atom, asvalency permits. A heterocyclyl group can either be monocyclic(“monocyclic heterocyclyl”) or a fused, bridged or spiro ring systemsuch as a bicyclic system (“bicyclic heterocyclyl”), and can besaturated or can be partially unsaturated. Heterocyclyl bicyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently optionally substituted, i.e.,unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a“substituted heterocyclyl”) with one or more substituents. In certainembodiments, the heterocyclyl group is unsubstituted 3-10 memberedheterocyclyl. In certain embodiments, the heterocyclyl group issubstituted 3-10 membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 memberedheterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8membered non-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-6 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-6 membered heterocyclyl”). In some embodiments, the 5-6 memberedheterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen,and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2ring heteroatoms selected from nitrogen, oxygen, and sulfur. In someembodiments, the 5-6 membered heterocyclyl has one ring heteroatomselected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing one heteroatominclude, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary4-membered heterocyclyl groups containing one heteroatom include,without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary5-membered heterocyclyl groups containing one heteroatom include,without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyland pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining two heteroatoms include, without limitation, dioxolanyl,oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-memberedheterocyclyl groups containing three heteroatoms include, withoutlimitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary6-membered heterocyclyl groups containing one heteroatom include,without limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl,and thianyl. Exemplary 6-membered heterocyclyl groups containing twoheteroatoms include, without limitation, piperazinyl, morpholinyl,dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containingtwo heteroatoms include, without limitation, triazinanyl. Exemplary7-membered heterocyclyl groups containing one heteroatom include,without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary8-membered heterocyclyl groups containing one heteroatom include,without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary5-membered heterocyclyl groups fused to a C₆ aryl ring (also referred toherein as a 5,6-bicyclic heterocyclic ring) include, without limitation,indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groupsfused to an aryl ring (also referred to herein as a 6,6-bicyclicheterocyclic ring) include, without limitation, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and the like.

Particular examples of heterocyclyl groups are shown in the followingillustrative examples:

wherein each W is selected from CR⁶⁷, C(R⁶⁷)₂, NR⁶⁷, O, and S; and eachY is selected from NR⁶⁷, O, and S; and R⁶⁷ is independently hydrogen,C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl. These heterocyclyl rings may be optionallysubstituted with one or more substituents selected from the groupconsisting of the group consisting of acyl, acylamino, acyloxy, alkoxy,alkoxycarbonyl, alkoxycarbonylamino, amino, substituted amino,aminocarbonyl (carbamoyl or amido), aminocarbonylamino, aminosulfonyl,sulfonylamino, aryl, aryloxy, azido, carboxyl, cyano, cycloalkyl,halogen, hydroxy, keto, nitro, thiol, —S-alkyl, —S-aryl, —S(O)-alkyl,—S(O)-aryl, —S(O)₂-alkyl, and —S(O)₂-aryl. Substituting groups includecarbonyl or thiocarbonyl which provide, for example, lactam and ureaderivatives.

“Hetero” when used to describe a compound or a group present on acompound means that one or more carbon atoms in the compound or grouphave been replaced by a nitrogen, oxygen, or sulfur heteroatom. Heteromay be applied to any of the hydrocarbyl groups described above such asalkyl, e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl, e.g.,heteroaryl, cycloalkenyl, e.g., cycloheteroalkenyl, and the like havingfrom 1 to 5, and particularly from 1 to 3 heteroatoms.

“Acyl” refers to a radical —C(O)R²⁰, where R²⁰ is hydrogen, substitutedor unsubstituted alkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted alkynyl, substituted or unsubstitutedcarbocyclyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, asdefined herein. “Alkanoyl” is an acyl group wherein R²⁰ is a group otherthan hydrogen. Representative acyl groups include, but are not limitedto, formyl (—CHO), acetyl (—C(═O)CH₃), cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl (—C(═O)Ph), benzylcarbonyl(—C(═O)CH₂Ph), —C(O)—C₁-C₈ alkyl, —C(O)—(CH₂)_(t)(C₆-C₁₀ aryl),—C(O)—(CH₂)_(t)(5-10 membered heteroaryl), —C(O)—(CH₂)_(t)(C₃-C₁₀cycloalkyl), and —C(O)—(CH₂)_(t)(4-10 membered heterocyclyl), wherein tis an integer from 0 to 4. In certain embodiments, R²¹ is C₁-C₈ alkyl,substituted with halo or hydroxy; or C₃-C₁₀ cycloalkyl, 4-10 memberedheterocyclyl, C₆-C₁₀ aryl, arylalkyl, 5-10 membered heteroaryl orheteroarylalkyl, each of which is substituted with unsubstituted C₁-C₄alkyl, halo, unsubstituted C₁-C₄ alkoxy, unsubstituted C₁-C₄ haloalkyl,unsubstituted C₁-C₄ hydroxyalkyl, or unsubstituted C₁-C₄ haloalkoxy orhydroxy.

“Acylamino” refers to a radical —NR²²C(O)R²³, where each instance of R²²and R23 is independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted carbocyclyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, as defined herein, or R²² is anamino protecting group. Exemplary “acylamino” groups include, but arenot limited to, formylamino, acetylamino, cyclohexylcarbonylamino,cyclohexylmethyl-carbonylamino, benzoylamino and benzylcarbonylamino.Particular exemplary “acylamino” groups are —NR²⁴C(O)—C₁-C₈ alkyl,—NR²⁴C(O)—(CH₂)_(t)(C₆-C₁₀ aryl), —NR²⁴C(O)—(CH₂)_(t)(5-10 memberedheteroaryl), —NR²⁴C(O)—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and—NR²⁴C(O)—(CH₂)_(t)(4-10 membered heterocyclyl), wherein t is an integerfrom 0 to 4, and each R²⁴ independently represents H or C₁-C₈ alkyl. Incertain embodiments, R²⁵ is H, C₁-C₈ alkyl, substituted with halo orhydroxy; C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl,arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which issubstituted with unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxy; and R²⁶ is H, C₁-C₈ alkyl,substituted with halo or hydroxy;

C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl, arylalkyl,5-10 membered heteroaryl or heteroarylalkyl, each of which issubstituted with unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxyl; provided that at leastone of R²⁵ and R²⁶ is other than H.

“Acyloxy” refers to a radical —OC(O)R²⁷, where R²⁷ is hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl, as defined herein. Representative examples include, but arenot limited to, formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl and benzylcarbonyl. In certainembodiments, R²⁸ is C₁-C₈ alkyl, substituted with halo or hydroxy;C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl, arylalkyl,5-10 membered heteroaryl or heteroarylalkyl, each of which issubstituted with unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxy.

“Alkoxy” refers to the group —OR²⁹ where R²⁹ is substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl. Particular alkoxygroups are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, and 1,2-dimethylbutoxy.Particular alkoxy groups are lower alkoxy, i.e. with between 1 and 6carbon atoms. Further particular alkoxy groups have between 1 and 4carbon atoms.

In certain embodiments, R²⁹ is a group that has 1 or more substituents,for instance, from 1 to 5 substituents, and particularly from 1 to 3substituents, in particular 1 substituent, selected from the groupconsisting of amino, substituted amino, C₆-C₁₀ aryl, aryloxy, carboxyl,cyano, C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, halogen, 5-10membered heteroaryl, hydroxyl, nitro, thioalkoxy, thioaryloxy, thiol,alkyl-S(O)—, aryl-S(O)—, alkyl-S(O)₂— and aryl-S(O)₂—. Exemplary‘substituted alkoxy’ groups include, but are not limited to,—O—(CH₂)_(t)(C₆-C₁₀ aryl), —O—(CH₂)_(t)(5-10 membered heteroaryl),—O—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —O—(CH₂)_(t)(4-10 memberedheterocyclyl), wherein t is an integer from 0 to 4 and any aryl,heteroaryl, cycloalkyl or heterocyclyl groups present, may themselves besubstituted by unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxy. Particular exemplary‘substituted alkoxy’ groups are —OCF₃, —OCH₂CF₃, —OCH₂Ph,—OCH₂-cyclopropyl, —OCH₂CH₂OH, and —OCH₂CH₂NMe₂.

“Amino” refers to the radical —NH₂.

“Substituted amino” refers to an amino group of the formula —N(R³⁸)₂wherein R³⁸ is hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted carbocyclyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, or an amino protecting group, wherein at leastone of R³⁸ is not a hydrogen. In certain embodiments, each R³⁸ isindependently selected from: hydrogen, C₁-C₈ alkyl, C₃-C₈ alkenyl, C₃-C₈alkynyl, C₆-C₁₀ aryl, 5-10 membered heteroaryl, 4-10 memberedheterocyclyl, or C₃-C₁₀ cycloalkyl; or C₁-C₈ alkyl, substituted withhalo or hydroxy; C₃-C₈ alkenyl, substituted with halo or hydroxy; C₃-C₈alkynyl, substituted with halo or hydroxy, or —(CH₂)_(t)(C₆-C₁₀ aryl),—(CH₂)_(t)(5-10 membered heteroaryl), —(CH₂)_(t)(C₃-C₁₀ cycloalkyl), or—(CH₂)_(t)(4-10 membered heterocyclyl), wherein t is an integer between0 and 8, each of which is substituted by unsubstituted C₁-C₄ alkyl,halo, unsubstituted C₁-C₄ alkoxy, unsubstituted C₁-C₄ haloalkyl,unsubstituted C₁-C₄ hydroxyalkyl, or unsubstituted C₁-C₄ haloalkoxy orhydroxy; or both R³⁸ groups are joined to form an alkylene group.

Exemplary ‘substituted amino’ groups are —NR³⁹—C₁-C₈ alkyl,—NR³⁹—(CH₂)_(t)(C₆-C₁₀ aryl), —NR³⁹—(CH₂)_(t)(5-10 membered heteroaryl),—NR³⁹—(CH₂)_(t)(C₃-C₁₀ cycloalkyl), and —NR³⁹—(CH₂)_(t)(4-10 memberedheterocyclyl), wherein t is an integer from 0 to 4, for instance 1 or 2,each R³⁹ independently represents H or C₁-C₈ alkyl; and any alkyl groupspresent, may themselves be substituted by halo, substituted orunsubstituted amino, or hydroxy; and any aryl, heteroaryl, cycloalkyl,or heterocyclyl groups present, may themselves be substituted byunsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄ alkoxy,unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl, orunsubstituted C₁-C₄ haloalkoxy or hydroxy. For the avoidance of doubtthe term ‘substituted amino’ includes the groups alkylamino, substitutedalkylamino, alkylarylamino, substituted alkylarylamino, arylamino,substituted arylamino, dialkylamino, and substituted dialkylamino asdefined below. Substituted amino encompasses both monosubstituted aminoand disubstituted amino groups.

“Azido” refers to the radical —N₃.

“Carbamoyl” or “amido” refers to the radical —C(O)NH₂.

“Substituted carbamoyl” or “substituted amido” refers to the radical—C(O)N(R⁶²)₂ wherein each R⁶² is independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, substituted or unsubstituted carbocyclyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, or an amino protectinggroup, wherein at least one of R⁶² is not a hydrogen. In certainembodiments, R⁶² is selected from H, C₁-C₈ alkyl, C₃-C₁₀ cycloalkyl,4-10 membered heterocyclyl, C₆-C₁₀ aryl, aralkyl, 5-10 memberedheteroaryl, and heteroaralkyl; or C₁-C₅ alkyl substituted with halo orhydroxy; or C₃-C₁₀ cycloalkyl, 4-10 membered heterocyclyl, C₆-C₁₀ aryl,aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of which issubstituted by unsubstituted C₁-C₄ alkyl, halo, unsubstituted C₁-C₄alkoxy, unsubstituted C₁-C₄ haloalkyl, unsubstituted C₁-C₄ hydroxyalkyl,or unsubstituted C₁-C₄ haloalkoxy or hydroxy; provided that at least oneR⁶² is other than H.

Exemplary ‘substituted carbamoyl’ groups include, but are not limitedto, —C(O) NR⁶⁴—C₁-C₈ alkyl, —C(O)NR⁶⁴—(CH₂)_(t)(C₆-C₁₀ aryl),—C(O)N⁶⁴—(CH₂)_(t)(5-10 membered heteroaryl), —C(O)NR⁶⁴—(CH₂)_(t)(C₃-C₁₀cycloalkyl), and —C(O)NR⁶⁴—(CH₂)_(t)(4-10 membered heterocyclyl),wherein t is an integer from 0 to 4, each R⁶⁴ independently represents Hor C₁-C₅ alkyl and any aryl, heteroaryl, cycloalkyl or heterocyclylgroups present, may themselves be substituted by unsubstituted C₁-C₄alkyl, halo, unsubstituted C₁-C₄ alkoxy, unsubstituted C₁-C₄ haloalkyl,unsubstituted C₁-C₄ hydroxyalkyl, or unsubstituted C₁-C₄ haloalkoxy orhydroxy.

‘Carboxy’ refers to the radical —C(O)OH.

“Cyano” refers to the radical —CN.

“Halo” or “halogen” refers to fluoro (F), chloro (Cl), bromo (Br), andiodo (I). In certain embodiments, the halo group is either fluoro orchloro. In further embodiments, the halo group is iodo.

“Hydroxy” refers to the radical —OH.

“Nitro” refers to the radical —NO₂.

“Cycloalkylalkyl” refers to an alkyl radical in which the alkyl group issubstituted with a cycloalkyl group. Typical cycloalkylalkyl groupsinclude, but are not limited to, cyclopropylmethyl, cyclobutylmethyl,cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl,cyclooctylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl,cyclohexylethyl, cycloheptylethyl, and cyclooctylethyl, and the like.

“Heterocyclylalkyl” refers to an alkyl radical in which the alkyl groupis substituted with a heterocyclyl group. Typical heterocyclylalkylgroups include, but are not limited to, pyrrolidinylmethyl,piperidinylmethyl, piperazinylmethyl, morpholinylmethyl,pyrrolidinylethyl, piperidinylethyl, piperazinylethyl, morpholinylethyl,and the like.

“Cycloalkenyl” refers to substituted or unsubstituted carbocyclyl grouphaving from 3 to 10 carbon atoms and having a single cyclic ring ormultiple condensed rings, including fused and bridged ring systems andhaving at least one and particularly from 1 to 2 sites of olefinicunsaturation. Such cycloalkenyl groups include, by way of example,single ring structures such as cyclohexenyl, cyclopentenyl,cyclopropenyl, and the like.

“Fused cycloalkenyl” refers to a cycloalkenyl having two of its ringcarbon atoms in common with a second aliphatic or aromatic ring andhaving its olefinic unsaturation located to impart aromaticity to thecycloalkenyl ring.

“Ethenyl” refers to substituted or unsubstituted —(C═C)—.

“Ethylene” refers to substituted or unsubstituted —(C—C)—.

“Ethynyl” refers to —(C≡C)—.

“Nitrogen-containing heterocyclyl” group means a 4- to 7-memberednon-aromatic cyclic group containing at least one nitrogen atom, forexample, but without limitation, morpholine, piperidine (e.g.2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g.2-pyrrolidinyl and 3-pyrrolidinyl), azetidine, pyrrolidone, imidazoline,imidazolidinone, 2-pyrazoline, pyrazolidine, piperazine, and N-alkylpiperazines such as N-methyl piperazine. Particular examples includeazetidine, piperidone and piperazone.

“Thioketo” refers to the group ═S.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylgroups, as defined herein, are optionally substituted (e.g.,“substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted”alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl group). In general, the term “substituted”,whether preceded by the term “optionally” or not, means that at leastone hydrogen present on a group (e.g., a carbon or nitrogen atom) isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₂, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(a), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa),—NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa),—S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(═O)(R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(ORC)₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂, —P(R^(cc))₃,—OP(R^(cc))₂, —OP(R^(cc))₃, —B(R^(aa))₂, —B(OR^(cc))₂,—BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc); each instance of R^(aa)is, independently, selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(aa) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups; each instance of R^(bb) is, independently, selectedfrom hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(a), —SO₂R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(c), —P(═O)₂R^(aa),P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(bb) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups; each instance ofR^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(cc) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff) ₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), C(═NR^(ff))N(R^(ff))₂,—OC(═NR)N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee),—SO₂N(R^(ee))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee), —S(═O)R^(ee),—Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂, —C(═O)SR^(ee),—C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee), —P(═O)(R^(ee))₂,—OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl,C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or twogeminal R^(dd) substituents can be joined to form ═O or ═S;each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀aryl, 3-10 membered heterocyclyl, and 3-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups;each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,3-10 membered heterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, ortwo R^(ff) groups are joined to form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups; and each instance ofR^(gg) is, independently, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH,—OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₃ ⁺X⁻,—NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃ ⁺X⁻, —N(OC₁₋₆alkyl)(C₁₋₆alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH, —SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl),—C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl), —OC(═O)(C₁₋₆ alkyl),—OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂, —OC(═O)NH(C₁₋₆alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)C(═O)(C₁₋₆alkyl),—NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂, —NHC(═O)NH(C₁₋₆ alkyl),—NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆ alkyl), —OC(═NH)OC₁₋₆alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆ alkyl), —C(═NH)NH₂,—OC(═NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl), —OC(NH)NH₂,—NHC(NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₆ alkyl,—OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —OSi(C₁₋₆alkyl)₃-C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂, —C(═O)S(C₁₋₆alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)₂(C₁₋₆ alkyl),—P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆ alkyl)₂, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or twogeminal R^(gg) substituents can be joined to form ═O or ═S; wherein X isa counterion.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a cationic quaternary amino group in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HSO₄ ⁻, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substitutents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂, —P(═O)(NR^(cc))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups attached to a nitrogen atom are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and R^(dd) are asdefined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa), R^(bb),R^(cc) and R^(dd) are as defined herein. Nitrogen protecting groups arewell known in the art and include those described in detail inProtecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts,3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamante, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), 3-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃, —P(═O)₂R^(aa), —P(═O)(R^(aa))₂,—P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and —P(═O)(NR^(bb))₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is ansulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —R^(aa),—N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—S(═O)R^(aa), —SO₂R^(aa), Si(R^(aa))₃, —P(R^(cc))₂, —P(R^(cc))₃,—P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂, and—P(═O)(NR^(bb))₂, wherein R^(aa), R^(bb), and R^(cc) are as definedherein. Sulfur protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

“Compounds of the present invention”, and equivalent expressions, aremeant to embrace the compounds as hereinbefore described, in particularcompounds according to any of the Formula herein recited and/ordescribed, which expression includes the prodrugs, the pharmaceuticallyacceptable salts, and the solvates, e.g., hydrates, where the context sopermits. Similarly, reference to intermediates, whether or not theythemselves are claimed, is meant to embrace their salts, and solvates,where the context so permits.

These and other exemplary substituents are described in more detail inthe Detailed Description, Examples, and claims. The invention is notintended to be limited in any manner by the above exemplary listing ofsubstituents.

Other Definitions

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government or thecorresponding agency in countries other than the United States, or thatis listed in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals, and more particularly, in humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound of theinvention that is pharmaceutically acceptable and that possesses thedesired pharmacological activity of the parent compound. In particular,such salts are non-toxic may be inorganic or organic acid addition saltsand base addition salts. Specifically, such salts include: (1) acidaddition salts, formed with inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and thelike; or formed with organic acids such as acetic acid, propionic acid,hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like; or (2)salts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, N-methylglucamine and thelike. Salts further include, by way of example only, sodium, potassium,calcium, magnesium, ammonium, tetraalkylammonium, and the like; and whenthe compound contains a basic functionality, salts of non toxic organicor inorganic acids, such as hydrochloride, hydrobromide, tartrate,mesylate, acetate, maleate, oxalate and the like. The term“pharmaceutically acceptable cation” refers to an acceptable cationiccounter-ion of an acidic functional group. Such cations are exemplifiedby sodium, potassium, calcium, magnesium, ammonium, tetraalkylammoniumcations, and the like (see, e.g., Berge, et al., J. Pharm. Sci. 66(1):1-79 (January ″77).

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient or carrier with which a compound of the invention isadministered.

“Pharmaceutically acceptable metabolically cleavable group” refers to agroup which is cleaved in vivo to yield the parent molecule of thestructural Formula indicated herein. Examples of metabolically cleavablegroups include —COR, —COOR, —CONRR and —CH₂OR radicals, where R isselected independently at each occurrence from alkyl, trialkylsilyl,carbocyclic aryl or carbocyclic aryl substituted with one or more ofalkyl, halogen, hydroxy or alkoxy. Specific examples of representativemetabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl,methoxymethyl and trimethylsilyl groups.

“Prodrugs” refers to compounds, including derivatives of the compoundsof the invention, which have cleavable groups and become by solvolysisor under physiological conditions the compounds of the invention thatare pharmaceutically active in vivo. Such examples include, but are notlimited to, choline ester derivatives and the like, N-alkylmorpholineesters and the like. Other derivatives of the compounds of thisinvention have activity in both their acid and acid derivative forms,but in the acid sensitive form often offers advantages of solubility,tissue compatibility, or delayed release in the mammalian organism (see,Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam1985). Prodrugs include acid derivatives well know to practitioners ofthe art, such as, for example, esters prepared by reaction of the parentacid with a suitable alcohol, or amides prepared by reaction of theparent acid compound with a substituted or unsubstituted amine, or acidanhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters,amides and anhydrides derived from acidic groups pendant on thecompounds of this invention are particular prodrugs. In some cases it isdesirable to prepare double ester type prodrugs such as (acyloxy)alkylesters or ((alkoxycarbonyl)oxy)alkylesters. Particularly the C₁ to C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aryl, C₇-C₁₂ substituted aryl, andC₇-C₁₂ arylalkyl esters of the compounds of the invention.

“Solvate” refers to forms of the compound that are associated with asolvent or water (also referred to as “hydrate”), usually by asolvolysis reaction. This physical association includes hydrogenbonding. Conventional solvents include water, ethanol, acetic acid andthe like. The compounds of the invention may be prepared e.g. incrystalline form and may be solvated or hydrated. Suitable solvatesinclude pharmaceutically acceptable solvates, such as hydrates, andfurther include both stoichiometric solvates and non-stoichiometricsolvates. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates and methanolates.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g., infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or anon-human animal, e.g., a mammal such as primates (e.g., cynomolgusmonkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents,cats, and/or dogs. In certain embodiments, the subject is a human. Incertain embodiments, the subject is a non-human animal. The terms“human”, “patient” and “subject” are used interchangeably herein.

“Therapeutically effective amount” means the amount of a compound that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” can vary depending on the compound, the disease and itsseverity, and the age, weight, etc., of the subject to be treated.

“Preventing” or “prevention” refers to a reduction in risk of acquiringor developing a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a subject not yetexposed to a disease-causing agent, or predisposed to the disease inadvance of disease onset.

The term “prophylaxis” is related to “prevention”, and refers to ameasure or procedure the purpose of which is to prevent, rather than totreat or cure a disease. Non-limiting examples of prophylactic measuresmay include the administration of vaccines; the administration of lowmolecular weight heparin to hospital patients at risk for thrombosisdue, for example, to immobilization; and the administration of ananti-malarial agent such as chloroquine, in advance of a visit to ageographical region where malaria is endemic or the risk of contractingmalaria is high.

“Treating” or “treatment” of any disease or disorder refers, in certainembodiments, to ameliorating the disease or disorder (i.e., arrestingthe disease or reducing the manifestation, extent or severity of atleast one of the clinical symptoms thereof). In another embodiment“treating” or “treatment” refers to ameliorating at least one physicalparameter, which may not be discernible by the subject. In yet anotherembodiment, “treating” or “treatment” refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In a further embodiment, “treating” or “treatment”relates to slowing the progression of the disease.

As used herein, the term “isotopic variant” refers to a compound thatcontains unnatural proportions of isotopes at one or more of the atomsthat constitute such compound. For example, an “isotopic variant” of acompound can contain one or more non-radioactive isotopes, such as forexample, deuterium (²H or D), carbon-13 (¹³C), nitrogen-15 (¹⁵N), or thelike. It will be understood that, in a compound where such isotopicsubstitution is made, the following atoms, where present, may vary, sothat for example, any hydrogen may be ²H/D, any carbon may be ¹³C, orany nitrogen may be ¹⁵N, and that the presence and placement of suchatoms may be determined within the skill of the art. Likewise, theinvention may include the preparation of isotopic variants withradioisotopes, in the instance for example, where the resultingcompounds may be used for drug and/or substrate tissue distributionstudies. The radioactive isotopes tritium, i.e., ³H, and carbon-14,i.e., ¹⁴C, are particularly useful for this purpose in view of theirease of incorporation and ready means of detection. Further, compoundsmay be prepared that are substituted with positron emitting isotopes,such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, and would be useful in Positron EmissionTopography (PET) studies for examining substrate receptor occupancy. Allisotopic variants of the compounds provided herein, radioactive or not,are intended to be encompassed within the scope of the invention.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

Stereoisomers that are not mirror images of one another are termed“diastereomers” and those that are non-superimposable mirror images ofeach other are termed “enantiomers”. When a compound has an asymmetriccenter, for example, when it is bonded to four different groups, a pairof enantiomers is possible. An enantiomer can be characterized by theabsolute configuration of its asymmetric center and is described by theR- and S-sequencing rules of Cahn and Prelog, or by the manner in whichthe molecule rotates the plane of polarized light and designated asdextrorotatory or levorotatory (i.e., as (+) or (−)-isomersrespectively). A chiral compound can exist as either individualenantiomer or as a mixture thereof. A mixture containing equalproportions of the enantiomers is called a “racemic mixture”.

“Tautomers” refer to compounds that are interchangeable forms of aparticular compound structure, and that vary in the displacement ofhydrogen atoms and electrons. Thus, two structures may be in equilibriumthrough the movement of π electrons and an atom (usually H). Forexample, enols and ketones are tautomers because they are rapidlyinterconverted by treatment with either acid or base. Another example oftautomerism is the aci- and nitro-forms of phenylnitromethane, which arelikewise formed by treatment with acid or base. Tautomeric forms may berelevant to the attainment of the optimal chemical reactivity andbiological activity of a compound of interest.

As used herein a pure enantiomeric compound is substantially free fromother enantiomers or stereoisomers of the compound (i.e., inenantiomeric excess). In other words, an “S” form of the compound issubstantially free from the “R” form of the compound and is, thus, inenantiomeric excess of the “R” form. The term “enantiomerically pure” or“pure enantiomer” denotes that the compound comprises more than 75% byweight, more than 80% by weight, more than 85% by weight, more than 90%by weight, more than 91% by weight, more than 92% by weight, more than93% by weight, more than 94% by weight, more than 95% by weight, morethan 96% by weight, more than 97% by weight, more than 98% by weight,more than 98.5% by weight, more than 99% by weight, more than 99.2% byweight, more than 99.5% by weight, more than 99.6% by weight, more than99.7% by weight, more than 99.8% by weight or more than 99.9% by weight,of the enantiomer. In certain embodiments, the weights are based upontotal weight of all enantiomers or stereoisomers of the compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure R-compound” refers to at least about 80% byweight R-compound and at most about 20% by weight S-compound, at leastabout 90% by weight R-compound and at most about 10% by weightS-compound, at least about 95% by weight R-compound and at most about 5%by weight S-compound, at least about 99% by weight R-compound and atmost about 1% by weight S-compound, at least about 99.9% by weightR-compound or at most about 0.1% by weight S-compound. In certainembodiments, the weights are based upon total weight of compound.

As used herein and unless otherwise indicated, the term“enantiomerically pure S-compound” or “S-compound” refers to at leastabout 80% by weight S-compound and at most about 20% by weightR-compound, at least about 90% by weight S-compound and at most about10% by weight R-compound, at least about 95% by weight S-compound and atmost about 5% by weight R-compound, at least about 99% by weightS-compound and at most about 1% by weight R-compound or at least about99.9% by weight S-compound and at most about 0.1% by weight R-compound.In certain embodiments, the weights are based upon total weight ofcompound.

In the compositions provided herein, an enantiomerically pure compoundor a pharmaceutically acceptable salt, solvate, hydrate or prodrugthereof can be present with other active or inactive ingredients. Forexample, a pharmaceutical composition comprising enantiomerically pureR-compound can comprise, for example, about 90% excipient and about 10%enantiomerically pure R-compound. In certain embodiments, theenantiomerically pure R-compound in such compositions can, for example,comprise, at least about 95% by weight R-compound and at most about 5%by weight S-compound, by total weight of the compound. For example, apharmaceutical composition comprising enantiomerically pure S-compoundcan comprise, for example, about 90% excipient and about 10%enantiomerically pure S-compound. In certain embodiments, theenantiomerically pure S-compound in such compositions can, for example,comprise, at least about 95% by weight S-compound and at most about 5%by weight R-compound, by total weight of the compound. In certainembodiments, the active ingredient can be formulated with little or noexcipient or carrier.

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof.

Unless indicated otherwise, the description or naming of a particularcompound in the specification and claims is intended to include bothindividual enantiomers and mixtures, racemic or otherwise, thereof. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art.

One having ordinary skill in the art of organic synthesis will recognizethat the maximum number of heteroatoms in a stable, chemically feasibleheterocyclic ring, whether it is aromatic or non aromatic, is determinedby the size of the ring, the degree of unsaturation and the valence ofthe heteroatoms. In general, a heterocyclic ring may have one to fourheteroatoms so long as the heteroaromatic ring is chemically feasibleand stable.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In certain aspects, provided herein are pharmaceutical compositionscomprising of a bolaamphiphile complex.

In further aspects, provided herein are novel nano-sized vesiclescomprising of bolaamphiphilic compounds.

In certain aspects, provided herein are novel bolaamphiphile complexescomprising one or more bolaamphiphilic compounds and a biologicallyactive compound.

In one embodiment, the biologically active compound is a compound activeagainst ALS. In another embodiment, the biologically active compound isan analgesic compound.

In further aspects, provided herein are novel formulations ofbiologically active compounds with one or more bolaamphiphilic compoundsor with bolaamhphile vesicles.

In another aspect, provided here are methods of delivering biologicallyactive drugs agents into animal or human brain. In one embodiment, themethod comprises the step of administering to the animal or human apharmaceutical composition comprising of a bolaamphiphile complex; andwherein the bolaamphiphile complex comprises one or more bolaamphiphiliccompounds and a compound active against ALS. In one particularembodiment, the biologically active compound is an analgesic compound.

In one embodiment, the bolaamphiphilic complex comprises onebolaamphiphilic compound. In another embodiment, the bolaamphiphiliccomplex comprises two bolaamphiphilic compounds.

In one embodiment, the bolaamphiphilic compound consists of twohydrophilic headgroups linked through a long hydrophobic chain. Inanother embodiment, the hydrophilic headgroup is an amino containinggroup. In a specific embodiment, the hydrophilic headgroup is a tertiaryor quaternary amino containing group.

In one particular embodiment, the bolaamphiphilic compound is a compoundaccording to formula I:

HG²-L¹-HG¹   I

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

each HG¹ and HG² is independently a hydrophilic head group; and

L¹ is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker;unsubstituted or substituted with C₁-C₂₀ alkyl, hydroxyl, or oxo.

In one embodiment, the pharmaceutically acceptable salt is a quaternaryammonium salt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is heteroalkylene, or heteroalkenyl linker comprising C,N, and O atoms; unsubstituted or substituted with C₁-C₂₀ alkyl,hydroxyl, or oxo.

In another embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is

—O-L²-C(O)—O—(CH₂)_(n4)—O—C(O)-L³-O—, or

—O-L²-C(O)—O—(CH₂)_(n5)—O—C(O)—(CH₂)_(n6)—,

-   -   and wherein each L² and L³ is C₄-C₂₀ alkenyl linker;        unsubstituted or substituted with C₁-C₈ alkyl or hydroxy;    -   and n4, n5, and n6 is independently an integer from 4-20.

In one embodiment, each L² and L³ is independently—C(R¹)—C(OH)—CH₂—(CH═CH)—(CH₂)_(n7)—; R¹ is C₁-C₈ alkyl, and n7 isindependently an integer from 4-20.

In another embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is —O—(CH₂)_(n1)—O—C(O)—(CH₂)_(n2)—C(O)—O—(CH₂)_(n3)—O—.

In another embodiment, with respect to the bolaamphiphilic compound offormula I, L¹ is

wherein:

-   -   each Z¹ and Z² is independently —C(R³)₂—, —N(R³)— or —O—;    -   each R^(1a), R^(1b), R³, and R⁴ is independently H or C₁-C₈        alkyl;    -   each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH, or        alkoxy;    -   each n8, n9, n11, and n12 is independently an integer from 1-20;    -   n10 is an integer from 2-20; and    -   each dotted bond is independently a single or a double bond.    -   and wherein each methylene carbon is unsubstituted or        substituted with C₁-C₄ alkyl; and each n1, n2, and n3 is        independently an integer from 4-20.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, the bolaamphiphilic compound is a compound according toformula II, III, IV, V, or VI:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each HG¹ and HG² is independently a hydrophilic head group;    -   each Z¹ and Z² is independently —C(R³)₂—, —N(R³)— or —O—;    -   each R^(1a), R^(1b), R³, and R⁴ is independently H or C₁-C₈        alkyl;    -   each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH,        alkoxy, or O-HG¹ or O-HG²;    -   each n8, n9, n11, and n12 is independently an integer from 1-20;    -   n10 is an integer from 2-20; and    -   each dotted bond is independently a single or a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each n9 and n11 is independently aninteger from 2-12. In another embodiment, n9 and n11 is independently aninteger from 4-8. In a particular embodiment, each n9 and n11 is 7 or11.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each n8 and n12 is independently 1, 2, 3,or 4. In a particular embodiment, each n8 and n12 is 1.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each R^(2a) and R^(2b) is independentlyH, OH, or alkoxy. In another embodiment, each R^(2a) and R^(2b) isindependently H, OH, or OMe. In another embodiment, each R^(2a) andR^(2b) is independently-O-HG¹ or O-HG². In a particular embodiment, eachR^(2a) and R^(2b) is OH.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each R^(1a) and R^(1b) is independentlyH, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl,n-heptyl, or n-octyl. In a particular embodiment, each R^(1a) and R^(1b)is independently n-pentyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each dotted bond is a single bond. Inanother embodiment, each dotted bond is a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, n10 is an integer from 2-16. In anotherembodiment, n10 is an integer from 2-12. In a particular embodiment, n10is 2, 4, 6, 8, 10, 12, or 16.

In one embodiment, with respect to the bolaamphiphilic compound offormula IV, R⁴ is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl,or isopentyl. In another embodiment, R⁴ is Me, or Et. In a particularembodiment, R⁴ is Me.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each Z¹ and Z² is independently C(R³)₂—,or —N(R³)—. In another embodiment, each Z¹ and Z² is independentlyC(R³)₂—, or —N(R³)—; and each R³ is independently H, Me, Et, n-Pr, i-Pr,n-Bu, i-Bu, sec-Bu, n-pentyl, or isopentyl. In a particular embodiment,R³ is H.

In one embodiment, with respect to the bolaamphiphilic compound offormula II, III, IV, V, or VI, each Z¹ and Z² is —O—.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, each HG¹ and HG² is independently selectedfrom:

wherein:

-   -   X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a), and        R^(5b) is independently H or substituted or unsubstituted C₁-C₂₀        alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl; each R^(5c) is        independently substituted or unsubstituted C₁-C₂₀ alkyl; each R⁸        is independently H, substituted or unsubstituted C₁-C₂₀ alkyl,        alkoxy, or carboxy;    -   m1 is 0 or 1; and    -   each n13, n14, and n15 is independently an integer from 1-20.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and each m1is 0.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and each m1is 1.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and eachn13 is 1 or 2.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, or IV, HG¹ and HG² are as defined above, and eachn14 and n15 is independently 1, 2, 3, 4, or 5. In another embodiment,each n14 and n15 is independently 2 or 3.

In one particular embodiment, the bolaamphiphilic compound is a compoundaccording to formula VIIa, VIIb, VIIc, or VIId:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5C) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.

In another particular embodiment, the bolaamphiphilic compound is acompound according to formula VIIIa, VIIIb, VIIIc, or VIIId:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5c) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.

In another particular embodiment, the bolaamphiphilic compound is acompound according to formula IXa, IXb, or IXc:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5C) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.

In another particular embodiment, the bolaamphiphilic compound is acompound according to formula Xa, Xb, or Xc:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof;wherein:

-   -   each X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a),        and R^(5b) is independently H or substituted or unsubstituted        C₁-C₂₀ alkyl or R^(5a) and R^(5b) may join together to form an N        containing substituted or unsubstituted heteroaryl, or        substituted or unsubstituted heterocyclyl;        -   each R^(5C) is independently substituted or unsubstituted            C₁-C₂₀ alkyl;        -   n10 is an integer from 2-20; and        -   each dotted bond is independently a single or a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each dotted bond is asingle bond. In another embodiment, each dotted bond is a double bond.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is an integerfrom 2-16.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is an integerfrom 2-12.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is 2, 4, 6, 8,10, 12, or 16.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each R^(5a), R^(5b),and R^(5c) is independently substituted or unsubstituted C₁-C₂₀ alkyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each R^(5a), R^(5b),and R^(5c) is independently unsubstituted C₁-C₂₀ alkyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, one of R^(5a),R^(5b), and R^(5c) is C₁-C₂₀ alkyl substituted with —OC(O)R⁶; and R⁶ isC₁-C₂₀ alkyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, two of R^(5a),R^(5b), and R^(5c) are independently C₁-C₂₀ alkyl substituted with—OC(O)R⁶; and R⁶ is C₁-C₂₀ alkyl. In one embodiment, R⁶ is Me, Et, n-Pr,i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl, n-heptyl, orn-octyl. In a particular embodiment, R⁶ is Me.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, one of R^(5a),R^(5b), and R^(5c) is C₁-C₂₀ alkyl substituted with amino, alkylamino ordialkylamino.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, two of R^(5a),R^(5b), and R^(5c) are independently C₁-C₂₀ alkyl substituted withamino, alkylamino or dialkylamino.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R^(5a), and R^(5b)together with the N they are attached to form substituted orunsubstituted heteroaryl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R^(5a), and R^(5b)together with the N they are attached to form substituted orunsubstituted pyridyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R^(5a), and R^(5b)together with the N they are attached to form substituted orunsubstituted monocyclic or bicyclic heterocyclyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is substituted orunsubstituted

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is

substituted with one or more groups selected from alkoxy, acetyl, andsubstituted or unsubstituted Ph.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is —NMe₂ or —N⁺Me₃.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is—N(Me)-CH₂CH₂—OAc or —N⁺(Me)₂-CH₂CH₂—OAc.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is a chitosanylgroup; and the chitosanyl group is a poly-(D)glucosaminyl group with MWof 3800 to 20,000 Daltons, and is attached to the core via N.

In one embodiment, the chitosanyl group is

and wherein each p1 and p2 is independently an integer from 1-400; andeach R^(7a) is H or acyl.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is a substance Phead group. In one embodiment, the substance P head group is boundthrough the co-amino group of lysine. In another embodiment, X is—NH—(CH2)₄—C(H)(NH-Pro-Arg)-NH-Pro-Gly-Gly-Phe-Phe-Gly-Leu-Met.

In one embodiment, with respect to the bolaamphiphilic compound offormula VIIa-VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is a headgroupcomprising NK1R antagonist.

In one embodiment, the NK1R antagonist is

I {1-[4-(1H-tetrazol-5-yl)butyl]indol-3-yl}carbonyl-Hyp-Nal-N(methyl)-Bz1, (Hyp = (R)-4-hydroxy-L-proline; Nal = 3-L-(β-naphthyl)-alanine) II CP-99,994 III(+)-[2R,3R,4R,8R,9(3′R)-2-{1-[1(3,5bis(tri-fluoromethyl)phenyl]ethyl)oxy}-4(-3carboxy-3-methylpiperidinlyl)-3-phenyl-methyltetra- hydropyran

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is a pharmaceutically acceptablesalt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is in a form of a quaternary salt.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is in a form of a quaternary saltwith pharmaceutically acceptable alkyl halide or alkyl tosylate.

In one embodiment, with respect to the bolaamphiphilic compound offormula I, II, III, IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc andXa-Xc, the bolaamphiphilic compound is any one of the bolaambphiliccompounds listed in Table 1.

In another specific aspect, provided herein are methods forincorporating biologically active drugs in the bolavesicles. In oneembodiment, the bolavesicle comprises one or more bolaamphilic compoundsdescribed herein.

In another specific aspect, provided herein are methods forbrain-targeted drug delivery using the bolavesicles incorporated withbiologically active drug.

In one particular embodiment, the biologically active drug iskyotorphine or enkephaline.

In one particular embodiment, the biologically active drug is irinotecan(CPT-11 or(S)-4,11-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxolH-pyrano[3′,4′:6,7]-indolizino[1,2-b]quinolin-9-yl-[1,4′bipiperidine]-1′-carboxylate).

In another specific aspect, provided herein are methods for deliveringkyotorphine and enkephaline to the brain.

In another specific aspect, provided herein are methods for deliveringCPT-11 to the brain.

In another specific aspect, provided herein are nano-particles,comprising one or more bolaamphiphilic compounds and kyotorphine orenkephaline. In one embodiment, the bolaamphiphilic compounds andkyotorphine or enkephaline are encapsulated within the nano-particle.

In another specific aspect, provided herein are nano-particles,comprising one or more bolaamphiphilic compounds and CPT-11.

In another specific aspect, provided herein are pharmaceuticalcompositions, comprising a nano-sized particle comprising one or morebolaamphiphilic compounds and kyotorphine, enkephaline, or CPT-11; and apharmaceutically acceptable carrier.

In another specific aspect, provided herein are methods for treatment ordiagnosis of diseases or disorders selected from ALS and relateddiseases using the nano-particles, pharmaceutical compositions orformulations of the present invention.

In another specific aspect, provided herein are methods for treatment ofpain using the nano-particles, pharmaceutical compositions orformulations of the present invention.

The Derivatives and Precursors disclosed can be prepared as illustratedin the Schemes provided herein. The syntheses can involve initialconstruction of, for example, vemonia oil or direct functionalization ofnatural derivatives by organic synthesis manipulations such as, but notlimiting to, epoxide ring opening. In those processes involving oxiranylring opening, the epoxy group is opened by the addition of reagents suchas carboxylic acids or organic or inorganic nucleophiles. Such ringopening results in a mixture of two products in which the new group isintroduced at either of the two carbon atoms of the epoxide moiety. Thisprovides beta substituted alcohols in which the substitution positionmost remote from the CO group of the main aliphatic chain of thevernonia oil derivative is arbitrarily assigned as position 1, while theneighboring substituted carbon position is designated position 2. Forsimplicity purposes only, the Derivatives and Precursors shown hereinmay indicate structures with the hydroxy group always at position 2 butthe Derivatives and Precursors wherein the hydroxy is at position 1 arealso encompassed by the invention. Thus, a radical of the formula—CH(OH)—CH(R)— refers to the substitution of —OH at either the carboncloser to the CO group, designated position 2 or to the carbon atposition 1. Moreover, with respect to the preparation of symmetricalbolaamphiphiles made via introducing the head groups through an epoxymoiety (e.g., as in vernolic acid) or a double bond (—C═C—) as in monounsaturated fatty acids (e.g., oleic acid) a mixture of three differentderivatives will be produced. In certain embodiments, vesicles areprepared using the mixture of unfractionated positional isomers. In oneaspect of this embodiment, where one or more bolas are prepared fromvernolic acid, and in which a hydroxy group as well as the head groupintroduced through an epoxy or a fatty acid with the head groupintroduced through a double bond (—C═C—), the bola used in vesiclepreparation can actually be a mixture of three different positionalisomers.

In other embodiments, the three different derivatives are isolated.Accordingly, the vesicles disclosed herein can be made from a mixture ofthe three isomers of each derivative or, in other embodiments, theindividual isomers can be isolated and used for preparation of vesicles.

Symmetrical bolaamphiphiles can form relatively stable self aggregatevesicle structures by the use of additives such as cholesterol andcholesterol derivatives (e.g., cholesterol hemisuccinate, cholesterololeyl ether, anionic and cationic derivatives of cholesterol and thelike), or other additives including single headed amphiphiles with one,two or multiple aliphatic chains such as phospholipids, zwitterionic,acidic, or cationic lipids. Examples of zwitterionic lipids arephosphatidylcholines, phosphatidylethanol amines and sphingomyelins.Examples of acidic amphiphilic lipids are phosphatidylglycerols,phosphatidylserines, phosphatidylinositols, and phosphatidic acids.Examples of cationic amphipathic lipids are diacyl trimethylammoniumpropanes, diacyl dimethylammonium propanes, and stearylamines cationicamphiphiles such as spermine cholesterol carbamates, and the like, inoptimum concentrations which fill in the larger spaces on the outersurfaces, and/or add additional hydrophilicity to the particles. Suchadditives may be added to the reaction mixture during formation ofnanoparticles to enhance stability of the nanoparticles by filling inthe void volumes of in the upper surface of the vesicle membrane.

Stability of nano vesicles according to the present disclosure can bedemonstrated by dynamic light scattering (DLS) and transmission electronmicroscopy (TEM). For example, suspensions of the vesicles can be leftto stand for 1, 5, 10, and 30 days to assess the stability of thenanoparticle solution/suspension and then analyzed by DLS and TEM.

The vesicles disclosed herein may encapsulate within their core theactive agent, which in particular embodiments is selected from peptides,proteins, nucleotides and or non-polymeric agents. In certainembodiments, the active agent is also associated via one or morenon-covalent interactions to the vesicular membrane on the outer surfaceand/or the inner surface, optionally as pendant decorating the outer orinner surface, and may further be incorporated into the membranesurrounding the core. In certain aspects, biologically active peptides,proteins, nucleotides or non-polymeric agents that have a net electriccharge, may associate ionically with oppositely charged headgroups onthe vesicle surface and/or form salt complexes therewith.

In particular aspects of these embodiments, additives which may bebolaamphiphiles or single headed amphiphiles, comprise one or morebranching alkyl chains bearing polar or ionic pendants, wherein thealiphatic portions act as anchors into the vesicle's membrane and thependants (e.g., chitosan derivatives or polyamines or certain peptides)decorate the surface of the vesicle to enhance penetration throughvarious biological barriers such as the intestinal tract and the BBB,and in some instances are also selectively hydrolyzed at a given site orwithin a given organ. The concentration of these additives is readilyadjusted according to experimental determination.

In certain embodiments, the oral formulations of the present disclosurecomprise agents that enhance penetration through the membranes of the GItract and enable passage of intact nanoparticles containing the drug.These agents may be any of the additives mentioned above and, inparticular aspects of these embodiment, include chitosan and derivativesthereof, serving as vehicle surface ligands, as decorations or pendantson the vesicles, or the agents may be excipients added to theformulation.

In other embodiments, the nanoparticles and vesicles disclosed hereinmay comprise the fluorescent marker carboxyfluorescein (CF) encapsulatedtherein while in particular aspects, the nanoparticle and vesicles ofthe present disclosure may be decorated with one or more of PEG, e.g.PEG2000-vemonia derivatives as pendants. For example, two kinds ofPEG-vemonia derivatives can be used: PEG-ether derivatives, wherein PEGis bound via an ether bond to the oxygen of the opened epoxy ring of,e.g., vernolic acid and PEG-ester derivatives, wherein PEG is bound viaan ester bond to the carboxylic group of, e.g., vernolic acid.

In other embodiments, vesicles, made from synthetic amphiphiles, as wellas liposomes, made from synthetic or natural phospholipids,substantially (or totally) isolate the therapeutic agent from theenvironment allowing each vesicle or liposome to deliver many moleculesof the therapeutic agent. Moreover, the surface properties of thevesicle or liposome can be modified for biological stability, enhancedpenetration through biological barriers and targeting, independent ofthe physico-chemical properties of the encapsulated drug.

In still other embodiments, the headgroup is selected from: (i) cholineor thiocholine, O-alkyl, N-alkyl or ester derivatives thereof; (ii)non-aromatic amino acids with functional side chains such as glutamicacid, aspartic acid, lysine or cysteine, or an aromatic amino acid suchas tyrosine, tryptophan, phenylalanine and derivatives thereof such aslevodopa (3,4-dihydroxy-phenylalanine) and p-aminophenylalanine; (iii) apeptide or a peptide derivative that is specifically cleaved by anenzyme at a diseased site selected from enkephalin, N-acetyl-ala-ala, apeptide that constitutes a domain recognized by beta and gammasecretases, and a peptide that is recognized by stromelysins; (iv)saccharides such as glucose, mannose and ascorbic acid; and (v) othercompounds such as nicotine, cytosine, lobeline, polyethylene glycol, acannabinoid, or folic acid.

In further embodiments, nano-sized particle and vesicles disclosedherein further comprise at least one additive for one or more oftargeting purposes, enhancing permeability and increasing the stabilitythe vesicle or particle. Such additives, in particular aspects, mayselected from from: (i) a single headed amphiphilic derivativecomprising one, two or multiple aliphatic chains, preferably twoaliphatic chains linked to a midsection/spacer region such as—NH—(CH₂)₂—N—(CH₂)₂—N—, or —O—(CH₂)₂—N—(CH₂)₂—O—, and a sole headgroup,which may be a selectively cleavable headgroup or one containing a polaror ionic selectively cleavable group or moiety, attached to the N atomin the middle of said midsection. In other aspects, the additive can beselected from among cholesterol and cholesterol derivatives such ascholesteryl hemisuccinate; phospholipids, zwitterionic, acidic, orcationic lipids; chitosan and chitosan derivatives, such as vernolicacid-chitosan conjugate, quaternized chitosan, chitosan-polyethyleneglycol (PEG) conjugates, chitosan-polypropylene glycol (PPG) conjugates,chitosan N-conjugated with different amino acids, carboxyalkylatedchitosan, sulfonyl chitosan, carbohydrate-branchedN-(carboxymethylidene) chitosan and N-(carboxymethyl) chitosan;polyamines such as protamine, polylysine or polyarginine; ligands ofspecific receptors at a target site of a biological environment such asnicotine, cytisine, lobeline, 1-glutamic acid MK801, morphine,enkephalins, benzodiazepines such as diazepam (valium) and librium,dopamine agonists, dopamine antagonists tricyclic antidepressants,muscarinic agonists, muscarinic antagonists, cannabinoids andarachidonyl ethanol amide; polycationic polymers such as polyethyleneamine; peptides that enhance transport through the BBB such as OX 26,transferrins, polybrene, histone, cationic dendrimer, synthetic peptidesand polymyxin B nonapeptide (PMBN); monosaccharides such as glucose,mannose, ascorbic acid and derivatives thereof; modified proteins orantibodies that undergo absorptive-mediated or receptor-mediatedtranscytosis through the blood-brain barrier, such as bradykinin B2agonist RMP-7 or monoclonal antibody to the transferrin receptor;mucoadhesive polymers such as glycerides and steroidal detergents; andCa²⁺ chelators. The aforementioned head groups on the additives designedfor one or more of targeting purposes and enhancing permeability mayalso be a head group, preferably on an asymmetric bolaamphiphile whereinthe other head group is another moiety, or the head group on both sidesof a symmetrical bolaamphiphile. In a further embodiment thebolaamphiphile head groups that comprise the vesicles membranes caninteract with the active agents to be encapsulated to be delivered in tothe brain and brain sites, and or other targeted sites, by ionicinteractions to enhance the % encapsulation via complexation and well aspassive encapsulation within the vesicles core. Further the formulationmay contain other additives within the vehicles membranes to furtherenhance the degree of encapsulation of the active agents by interactionsother than ionic interactions such as polar or hydrophobic interactions.

In other embodiments, nano-sized particle and vesicles discloser hereinmay comprises at least one biologically active agent is selected from:(i) a natural or synthetic peptide or protein such as analgesicspeptides from the enkephalin class, insulin, insulin analogs, oxytocin,calcitonin, tyrotropin releasing hormone, follicle stimulating hormone,luteinizing hormone, vasopressin and vasopressin analogs, catalase,interleukin-II, interferon, colony stimulating factor, tumor necrosisfactor (TNF), melanocyte-stimulating hormone, superoxide dismutase,glial cell derived neurotrophic factor (GDNF) or the Gly-Leu-Phe (GLF)families; (ii) nucleosides and polynucleotides selected from DNA or RNAmolecules such as small interfering RNA (siRNA) or a DNA plasmid; (iii)antiviral and antibacterial; (iv) antineoplastic and chemotherapy agentssuch as cyclosporin, doxorubicin, epirubicin, bleomycin, cisplatin,carboplatin, vinca alkaloids, e.g. vincristine, Podophyllotoxin,taxanes, e.g. Taxol and Docetaxel, and topoisomerase inhibitors, e.g.irinotecan, topotecan.

Additional embodiments within the scope provided herein are set forth innon-limiting fashion elsewhere herein and in the examples. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting in any manner.

Pharmaceutical Compositions

In another aspect, the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of a compound of Formula I or a complex thereof.

When employed as pharmaceuticals, the compounds provided herein aretypically administered in the form of a pharmaceutical composition. Suchcompositions can be prepared in a manner well known in thepharmaceutical art and comprise at least one active compound.

In certain embodiments, with respect to the pharmaceutical composition,the carrier is a parenteral carrier, oral or topical carrier.

The present invention also relates to a compound or pharmaceuticalcomposition of compound according to Formula I; or a pharmaceuticallyacceptable salt or solvate thereof for use as a pharmaceutical or amedicament.

Generally, the compounds provided herein are administered in atherapeutically effective amount. The amount of the compound actuallyadministered will typically be determined by a physician, in the lightof the relevant circumstances, including the condition to be treated,the chosen route of administration, the actual compound administered,the age, weight, and response of the individual patient, the severity ofthe patient's symptoms, and the like.

The pharmaceutical compositions provided herein can be administered by avariety of routes including oral, rectal, transdermal, subcutaneous,intravenous, intramuscular, and intranasal. Depending on the intendedroute of delivery, the compounds provided herein are preferablyformulated as either injectable or oral compositions or as salves, aslotions or as patches all for transdermal administration.

The compositions for oral administration can take the form of bulkliquid solutions or suspensions, or bulk powders. More commonly,however, the compositions are presented in unit dosage forms tofacilitate accurate dosing. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. Typical unitdosage forms include prefilled, premeasured ampules or syringes of theliquid compositions or pills, tablets, capsules or the like in the caseof solid compositions. In such compositions, the compound is usually aminor component (from about 0.1 to about 50% by weight or preferablyfrom about 1 to about 40% by weight) with the remainder being variousvehicles or carriers and processing aids helpful for forming the desireddosing form.

Liquid forms suitable for oral administration may include a suitableaqueous or nonaqueous vehicle with buffers, suspending and dispensingagents, colorants, flavors and the like. Solid forms may include, forexample, any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

Injectable compositions are typically based upon injectable sterilesaline or phosphate-buffered saline or other injectable carriers knownin the art. As before, the active compound in such compositions istypically a minor component, often being from about 0.05 to 10% byweight with the remainder being the injectable carrier and the like.

Transdermal compositions are typically formulated as a topical ointmentor cream containing the active ingredient(s), generally in an amountranging from about 0.01 to about 20% by weight, preferably from about0.1 to about 20% by weight, preferably from about 0.1 to about 10% byweight, and more preferably from about 0.5 to about 15% by weight. Whenformulated as a ointment, the active ingredients will typically becombined with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredients may be formulated in a cream with,for example an oil-in-water cream base. Such transdermal formulationsare well-known in the art and generally include additional ingredientsto enhance the dermal penetration of stability of the active ingredientsor the formulation. All such known transdermal formulations andingredients are included within the scope provided herein.

The compounds provided herein can also be administered by a transdermaldevice. Accordingly, transdermal administration can be accomplishedusing a patch either of the reservoir or porous membrane type, or of asolid matrix variety.

The above-described components for orally administrable, injectable ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, MackPublishing Company, Easton, Pa., which is incorporated herein byreference.

The above-described components for orally administrable, injectable, ortopically administrable compositions are merely representative. Othermaterials as well as processing techniques and the like are set forth inPart 8 of Remington's The Science and Practice of Pharmacy, 21stedition, 2005, Publisher: Lippincott Williams & Wilkins, which isincorporated herein by reference.

The compounds of this invention can also be administered in sustainedrelease forms or from sustained release drug delivery systems. Adescription of representative sustained release materials can be foundin Remington's Pharmaceutical Sciences.

The present invention also relates to the pharmaceutically acceptableformulations of compounds of Formula I. In certain embodiments, theformulation comprises water. In another embodiment, the formulationcomprises a cyclodextrin derivative. In certain embodiments, theformulation comprises hexapropyl-β-cyclodextrin. In a more particularembodiment, the formulation comprises hexapropyl-β-cyclodextrin (10-50%in water).

The present invention also relates to the pharmaceutically acceptableacid addition salts of compounds of Formula I. The acids which are usedto prepare the pharmaceutically acceptable salts are those which formnon-toxic acid addition salts, i.e. salts containing pharmacologicallyacceptable aniovs such as the hydrochloride, hydroiodide, hydrobromide,nitrate, sulfate, bisulfate, phosphate, acetate, lactate, citrate,tartrate, succinate, maleate, fumarate, benzoate, para-toluenesulfonate,and the like.

The following formulation examples illustrate representativepharmaceutical compositions that may be prepared in accordance with thisinvention. The present invention, however, is not limited to thefollowing pharmaceutical compositions.

Formulation 1—Injection

A compound of the invention may be dissolved or suspended in a bufferedsterile saline injectable aqueous medium to a concentration ofapproximately 5 mg/mL.

Methods of Treatment

Bolaamphiphilic vesicles (bolavesicles) may have certain advantages overconventional liposomes as potential vehicles for drug delivery.Bolavesicles have thinner membranes than comparable liposomal bilayer,and therefore possess bigger inner volume and hence higher encapsulationcapacity than liposomes of the same diameter. Moreover, bolavesicles aremore physically-stable than conventional liposomes, but can bedestabilized in a triggered fashion (e.g., by hydrolysis of theheadgroups using a specific enzymatic reaction) thus allowing controlledrelease of the encapsulated material at the site of action (i.e., drugtargeting).

Thus, various biologically active drug molecules can be encapsulated inthe bolaamphiphilic vesicles and then delivered to the brain insufficient concentrations for therapeutic use.

The bola vesicles aggregate into encapsulating monolayer membraneswhich, together with functional surface groups, provide vesiclestability, penetrability through the BBB and a controlled releasemechanism that enables the release of the encapsulated drug primarily inthe brain.

The novel nanovesicles can encapsulates drugs, gets through theblood-brain barrier (BBB) and releases the drug in the brain. A majorfactor limiting the efficacy of some chemotherapeutical agents that arepotentially effective in the treatment of malignant gliomas,particularly glioblastoma multiforme (GBM), is that most drugs cannotcross the BBB. A study from Duke University showed that, out of 40 drugstested, CPT-11 (Irinotecan, used for solid tumors) was the most potentchemotherapeutic agent against patients' gliomas implanted in mice, andwas effective against every tumor tested.

However, attempts to treat GBM patients with CPT-11 were unsuccessfulbecause very little gets through the BBB and reaches the tumor. Hence,CPT-11 encapsulated within bola vesicles, can penetrate the brain viathe intense capillary network that supplies blood to the brain and canrelease CPT-11 upon reaching tumor cells. Thus, it would be effective intreating GBM. The efficacy of CPT-11 delivered by bola vesicles may befurther increased by administering it with oral temozolamide which, incombination with radiotherapy, prolongs survival by months and, based onliterature, acts synergistically with CPT-11 to kill gliomas.

In still further embodiments, the present disclosure also provides nanovesicles prepared from bolaamphiphilic compounds comprising encapsulatedcyclodextrin derivatives, as well as compositions comprising same anduses thereof.

More specifically, the present disclosure is directed to encapsulationof cyclodextrins within the aqueous core of the bolaamphiphilic vesiclesdescribed herein, which are designed to be administered systematically(e.g., intravenous, Intraperitoneal injection (IP) and oral) anddelivery the drug or active agents into he CNS/brain and spinal chord.

Three illustrative aspects of these embodiments include: 1) delivery ofempty cyclodextrins and cyclodextrins derivatives by bolaamphiphilevesicles to the brain (CNS) after systemic administration for thetreatment of Niemann-Pick type C disease; (2) selective delivery to thebrain (CNS) or spinal cord hydrophobic/lipophilic drugs or active agentswith low water solubility by the encapsulation of the said drug oractive agent in cyclodextrin or cyclodextrins derivatives which are thenencapsulated within bolaamphiphile vesicles with the characteristicsneeded to deliver the drug or active agents into the CNS/brain or spinalcord via systemic administration. In this way the total amount of drugor active agent per vesicle is increased as the active agent is not onlyencapsulated within the lipophilic membrane of the vesicle but alsowithin the vesicle core which contains the water soluble cyclodextrinswithin the hydrophobic cavity of cyclodextrins thehydrophobic/lipophilic active agent is encapsulated; and (3) in oneaspect, embodiment 2 is used to delivery calcium channel blockers andactivators to the CNS and spinal cord. Calcium channel blockers andactivators are often low or non water soluble and their delivery to theCNS is problematic as either they cannot penetrate the CNS and/or arelatively high concentrations must be systematically administered whichcauses significant toxic side effects. As described herein, calciumchannel blockers and activators are encapsulated in the bolaamphiphilevesicle which can efficiently delivery the active agent or drug into theCNS, via encapsulation in bolaamphiphile membrane and the withincyclodextrins derivatives which are encapsulated within the core of thevesicles and the cyclodextrins is hydrophilic on its external surfaces.Thus the therapeutic dose is reduced and the toxic effective reducedbecause of targeting to the brain organ by the vesicles whichefficiently deliver a high therapeutic dose of the calcium channelblocker or activator to the target site.

The present disclosure describes use of the cyclodextrin derivativehexapropyl-beta-cyclodextrin, and further relates to thepharmaceutically acceptable formulations of compounds of Formula I. Incertain embodiments, the cyclodextrin is embedded onto to the surface ofthe vesicles and it is anchored into the vesicle membrane through thehexylpropyl moiety; i.e., it is not encapsulated within the vesicle corerather attached to the surface, which may block the vesicle's ability topenetrate through biological organs and the amount of agent encapsulatedis limited as the amount of cyclodextrin groups on the surface issignificantly less than can be encapsulated with the core of thevesicle. Accordingly, the present disclosure further provides approachesfor encapsulating the cyclodextrins in the core of the vesicles.

Cyclodextrins are a family of compounds made up of sugar molecules boundtogether in a ring. The exterior of the ring is hydrophilic and theinterior is relatively hydrophobic. In this way the solubility ofmolecules with that have low water solubility can be improved by theirencapsulation within the cyclodextrin ring. They are used in food,pharmaceutical, drug delivery and chemical industries, as well asagriculture and environmental engineering. Cyclodextrins are composed of5 or more α-D-glucopyranoside units linked 1->4, as in amylose. Typicalcyclodextrins contain a number of glucose monomers ranging from six toeight units in a ring, creating a cone shape: (a) Alpha-cyclodextrin:6-membered sugar ring molecule; (b) β (beta)-cyclodextrin: 7-memberedsugar ring molecule, (c) γ (gamma)-cyclodextrin: 8-membered sugar ringmolecule, (d) Hydroxypropyl-β-cyclodextrin (HPβCD) and (e)Methyl-β-cyclodextrin. Each of these are encompassed within the presentdisclosure; also included are other cyclodextrin derivatives known inthe art, which may be incorporated with the bolaamphiphilic vesicles ofthe present invention. In particular for the treatment or prevention ofcertain diseases that require the removal of cholesterol a preferredembodiment of the present invention is the encapsulation ofβ-cyclodextrin and methyl-β-cyclodextrin (MβCD). Both β-cyclodextrin andmethyl-β-cyclodextrin (MβCD) can remove cholesterol from cultured cells.The methylated form MβCD was found to be more efficient thanβ-cyclodextrin. The water-soluble MβCD is known to form solubleinclusion complexes with cholesterol, thereby enhancing its solubilityin aqueous solution. MβCD is employed for the preparation ofcholesterol-free products: the bulky and hydrophobic cholesterolmolecule is easily lodged inside cyclodextrin rings that are thenremoved. MβCD is also employed in research to disrupt lipid rafts byremoving cholesterol from membranes. It has also been shown howcyclodextrin assists in moving cholesterol out of lysosomes inNiemann-Pick type C disease and thus treating this disease—Which is alysosomal storage disease causing progressive deterioration of thenervous system and dementia. It usually affects young children byinterfering with their ability to metabolize cholesterol at the cellularlevel. Numerous research studies have followed showing thathydroxypropyl-β-cyclodextrin (HPβCD) is not simply an agent tosolubilize drugs but has powerful pharmacological properties.

It is however difficult to get high concentrations of bothβ-cyclodextrin and methyl-β-cyclodextrin (MβCD) into he CNS to treatNiemann-Pick type C disease after systemic administration because oflimit biological stability in the blood and poor penetration through theblood brain barrier (BBB). Also, the delivery of empty cyclodextrins isof low efficiency as lipids and cholesterol and other lipophilicmolecules found in the blood and cell membranes can fill thecyclodextrin core and reducing the number of empty cyclodextrinsreaching the disease site. One approach to overcoming these issues wouldbe to cyclodextrins in the design of novel drug delivery with liposomes,which are limited as they are made from single headed phospholipids.Encapsulating the cyclodextrins within the liposomes results in thecyclodextrins extracting phospholipids and the cholesterol andcholesterol derivative additives used to form the liposomal membrane.Thus limiting the liposomes shelf life and biological stability. And inaddition much of the efficacy of the cyclodextrins is lost by thefilling of its internal hydrophobic core by the phospholipids andcholesterol additives.

The present inventors have surprising found that with the use ofbolaamphiphiles of specific molecular design, vesicles with encapsulatedempty cyclodextrins can be prepared such that the vesicles which can beused to deliver the cyclodextrins to the CNDs or spinal cord aftersystemic administration are stable and do not fill the hydrophobic coreof the cyclodextrin with the vesicles components. The bolaamphiphilesused in forming the vesicles have two relatively large ionic head groupsvesicles to prevent bolas filling interior of the cyclodextrins. Thusthe vesicle's bolaamphiphiles molecular structure with two “large”terminal hydrophilic head groups will prevent their uptake within thecyclodextrin ensuring vesicle stability and cyclodextrin efficacy. Thecyclodextrins water solubility allows for high therapeuticconcentrations in the aqueous core of our vesicles, and its interactionswith the interior bolaamphiphile head groups that comprise the vesiclesmembranes further enhancing loading within the vesicle.

It has also been discovered the inventors' bolaamphiphilic (bola)vesicles can be used to encapsulate cyclodextrins (CDs) withencapsulated hydrophobic or low water soluble drugs. This combinationstakes advantage of inventors' bola vesicles to delivery drugs to targetsites and organs such as the brains and specific sites within the brainand the high encapsulation efficiency that can be achieved for low watersoluble drugs which are encapsulated with this invention both within thebola membrane and within the water core of the vesicle by the watersoluble CD contain the active agent within its hydrophobic interior.

In contrast, liposomes entrap hydrophilic drugs in the aqueous phase andhydrophobic drugs in the lipid bilayers and retain drugs en route totheir destination. Major problems encountered with these vesicularsystems appears during their preparation and results from a low watersolubility of the drug is rapidly released in the presence of plasmaleading to either a low yield in drug loading, or a slow or incompleterelease rate of the drug. These limitations are overcome using thepresently-described approach involving entrapping the CD-drug complexesinto vesicles, which combines the advantages of both CDs (such asincreasing the solubility of drugs) and liposomes (such as targeting ofdrugs) into a single system and thus circumvents the problems associatedwith each system. By forming water soluble complexes, CDs would allowinsoluble drugs to accommodate in the aqueous phase of vesicles and thuspotentially increase drug-to-lipid mass ratio levels, enlarge the rangeof insoluble drugs amenable for encapsulation (i.e.,membrane-destabilizing agents), allow drug targeting, and reduce drugtoxicity.

Potential problems associated with intravenous administration of CDcomplexes (such as their rapid removal into urine and toxicity tokidneys, especially after chronic use), can be circumvented by theirentrapment in liposomes. Liposomal entrapment can also alter thepharmacokinetics of inclusion complexes. Liposomal entrapmentdrastically reduced the urinary loss of HP-b-CD/drug complexes butaugmented the uptake of the complexes by liver and spleen, where afterliposomal disintegration in tissues, drugs were metabolized at ratesdependent on the stability of the complexes.

Liposome's drug delivery systems are however not efficient activetargeting drug delivery systems because of their relatively poor intactpenetration through biological barriers, lack of stability needed for anactive delivery into specific organs and to sites within these organsand the inability to combine stability with an efficient releasemechanism at the target site. The bola vesicles of the presentdisclosure can achieve these objectives using bolas with specificmolecular structures that with other components that can self-assembleinto multifunctional particles with a high encapsulation efficiency,biological stability and intact penetration through biological barriers,targeting and an efficient disruption mechanism at the target site. Incombining these properties with the encapsulation of CD containing ahydrophobic or low water soluble agent or drug we can achieve a veryhigh encapsulation loading and efficient targeting to a given site ofthe encapsulated drug.

In one embodiment, calcium channel blockers and activators are deliveredto the CNS and spinal cord. Calcium (Ca) channel blockers and activatorsare often non water soluble and their delivery to the CNS is problematicas either they cannot penetrate the CNS and/or a relatively highconcentrations must be systematically administered which causessignificant toxic side effects. The present disclosure describesencapsulation of calcium channel blockers and activators in thebolaamphiphile vesicles which can efficiently delivery the active agentor drug into the CNS, via encapsulation in bolaamphiphile membrane andthe within cyclodextrins derivatives which are encapsulated within thecore of the vesicles and the cyclodextrins is hydrophilic. Thus thetherapeutic dose is reduced and the toxic effective reduced because oftargeting to the brain organ by the vesicles which efficiently deliver ahigh therapeutic dose of the calcium channel blocker or activator to thetarget site.

The different Ca channel blockers and activators that we can delivery tothe CNS are often used for treating non CNS diseases but have beneficialeffects on CNs diseases. Examples of such active agents include:

-   -   L-type are Ca channel blocker drugs are used as cardiac        antiarrhythmics or antihyoertensives, depending on whether the        drugs have higher affinity for the heart (the phenylalkylamines,        like verapamil), or for the vessels (the dihydropyridines, like        nifedipine). Calcium-channels, blockers have an established role        in the management of cardiac arrhythmias. They were identified        empirically with the idea of achieving selective inhibition of        voltage-gated calcium-channels and vasodilatation    -   Ca Channel control agents for the treatment of an amyloidosis        such as Alzheimer's disease comprises administering an inhibitor        of the interaction between A.beta. globulomer and the P/Q type        voltage-gated presynaptic calcium channel to said subject    -   Nitrone-based compositions for the prevention and treatment of a        variety of ophthalmic diseases or conditions where RPE65 protein        isomerohydrolase is implicated.    -   Nimodipine—is a dihydropyridine Ca channel blocker originally        developed for the treatment of high blood pressure.    -   Calcium channel blockers (calcium antagonists) have been used in        an attempt to prevent cerebral vasospasm after injury, maintain        blood flow to the brain, and so prevent further damage.    -   Ca channel active agents tested for the reduction of Parkinson's        disease risk that include isradipine, nimodipine, and        nifedipine, among others. All are dihydropyridine derivatives,        which block so-called L-type calcium channels on smooth muscle,        reducing the force of contraction and thus reducing blood        pressure. Amlodipine, doesn't readily cross the blood-brain        barrier was not evaluated by other but in our encapsulated in        our vesicles would readily cross the BBB into the brain and was        effective. In particular “the at risk of developing Parkinson's        disease should benefit by the use of calcium blockers such as        isradipine as it appears that the dopamine-producing cells in        the SN begin to disappear well before the onset of symptoms. By        the use of our vesicles combined with CD encapsulation we can        readily target our vesicles to the regions affect by Parkinson's        disease and thus have a highly beneficial effect.    -   Calcium channel blockers protect neurons by lowering blood        pressure and reversing cellular-level calcium channel        dysfunction that occurs with age, cerebral infarction, and        Alzheimer's disease (AD). The following illustrative Calcium        channel blockers can be used in the methods and compositions of        the present disclosure: (a) Select dihydropyridines inhibit        amyloidogenesis in apolipoprotein E carriers, such as,        amlodipine and nilvadipine reduce β-secretase activity and        amyloid precursor protein-β production; nilvadipine and        nitrendipine limit β-amyloid protein synthesis in the brain and        promote their clearance through the blood-brain barrier;        nilvadipine-treated apolipoprotein E carriers experience        cognitive stabilization compared with cognitive decreases seen        in non-treated subjects; (b) Dihydropyridines can produce        therapeutic effects for both AD and cerebrovascular dementia        patients, indicating the potential that certain agents in this        class have for treating both conditions.

In still further embodiments, the present disclosure also providesembodiments involving forming bola vesicles with a solid particle of ahydrophobic drug comprises dissolving one or more of the boladerivatives and other additives and the water insoluble drug in a watermiscible common solvent or solvent combination, and injecting it into anexcess of water such that the drug particles precipate out as nanoparticles within the core of the bola vesicles which self-assemblearound the precipitating drug. The “common solvent” refers to a solventor combination of solvents in which both the amphiphile and thehydrophobic drug dissolve.

In one embodiment, the common solvent is an alkanol such as ethanol orisopropyl alcohol, and the method consists in injecting the alcoholicsolution comprising the bola amphiphile and additives and thehydrophobic drug under the surface of an aqueous solvent, whereby thebola amphiphile forms vesicles within the encapsulated space of the bolavesicle the drug precipitates. Typically, this can be achieved byinjection of an alcoholic solution through a small bore Hamilton syringeinto a well-stirred aqueous solution. In addition to ethanol andisopropyl alcohol, other water-soluble alcohols and water-misciblesolvents such as tetrahydrofuran (THF), N-methylpyrrolidone (NMP),dimethylformamide (DMF) and dimethyl sulfoxide (DMSO), or a combinationthereof, may be used. The amount of solvent in the aqueous phase shouldbe sufficiently low so as to not disrupt the formed bola vesicles.

The hydrophobic drugs may be from many different categories and in oneembodiment these drugs are taken from Ca channel blockers and oractivators including those disclosed herein.

An example of the approach is to: Bolaamphiphiles (GLH 19 and GLH 20 ina ratio of 2/1), cholesterol, and CHEMS (2:1:1 mole ratio) where in thebolas are together at 20 mg and a calcium channel blocker are Amlodipine(20 mg) dissolved in 1 ml ethanol/DMSO at a ratio of ½. One ml ofnitrogen-purged aqueous media (e.g. water, saline, solute solution, etc)was placed in a 5 ml vial and stirred rapidly using a magnetic stirrer.A fine gauge needle was fitted to a 1 ml glass syringe and used to drawup to 100 .mu.l of the bola drug solution. The tip of the needle waspositioned below the surface of the stirred aqueous solution, and thebola d solution was injected as rapidly as possible into the aqueousmedia which was kept at room temperature. The bola vesicles were formedimmediately with encapsulated solid particles of drug.

The present disclosure further provides (a) surface-targeting mechanismscomprising the use of a tumor specific ligand to target vesicles tobrain tumor, (b) membrane release mechanisms involving the design headgroups hydrolyzable by Acetyl Cholinesterase (AChE, which is found athigh levels outside of GBM cells), (c) core-drug encapsulation,involving loading vesicles with chemotherapeutic that have provenpotency against human GBM, but no BBB permeability, (d) administrationmechanisms including intravenous and oral routes; and combinationtherapies.

In other embodiments, the present disclosure provides nano-sizedparticles comprising multi-headed amphiphiles for targeted drugdiscovery. In one aspect of such embodiments, that present disclosureprovides treatment of brain tumors by IV and oral administration,surface ligands on the vesicle surface for targeting to sites in thebrain, release mechanism form the vesicles with acetyl choline groups byacetyl choline esterase, use of surface ligands such as chitosan forenhancing penetration through the BBB, and GI tract. Vesicles useful inthese embodiments may comprise, e.g., cholesterol and cholesterolhemisuccinate, and chitosan alkyl conjugates to place chitosan surfacegroups on the vesicles' surface; such vesicles may comprise bolas withchitosan head groups and/or bola conjugates.

In specific aspects of these embodiments, the present disclosure alsoprovides targeting ligands, including the four illustrative ligandsdescribed below.

In one aspect, these embodiments include the synthesis of bolas withNK1R-ligand head groups, i.e., GBM tumor cells highly express theneurokinin-1-receptor (NK1R). Accordingly, such tumors are targeted byattaching NK1R ligands to the bola skeletons as head groups. The headgroups may be substance P, an endogenous peptide that serves as thenatural ligand for NK1R, and/or antagonists with high affinity to NK1R.These bolas are used as one of the building blocks in vesicleformulation to decorate the outer surface of the vesicle with atargeting ligand. A substance P-radiolabelled-analog has shown excellenttargeting of GBM tumors in patients.

Synthesis of bolas with substance P as the head group is achieved bycovalent binding of substance P to fatty acids using standard proteinconjugation methodologies, e.g., activation of the carboxylate byN-hydroxy succinimide in the presence of dicyclohexylcarboiimide andsubsequent formation of the amidic linkage. The aliphatic-amideproducts, which are formed, are very stable. For example, selectivederivatization of substance P peptide's lysine ω-amino group (asdepicted in the scheme below) may be used, since lysine amines arereasonably good nucleophiles above pH 8.0 (pKa=9.18) and react easilywith a variety of reagents to form stable bonds, while other aminogroups in the peptide are less reactive under these conditions. Fattyacid-substance P conjugates with variations in chain length andsaturation of the fatty acid moiety are also synthesized and examined todetermine their toxicity and ability to be incorporated into thevesicles.

The following provides an illustrative approach for synthesis of anamphiphilic compound with substance P head group bound through theω-amino group of lysine:

In a second approach, bolas with NK-1-receptor antagonist head groups asthe targeting ligand can be synthesized as well as bolas with NK1Rantagonists as the targeting ligand can be synthesized. The NK1Rantagonists, Peptide I and non-peptide compounds II and III (see below),are used. For the synthesis of the bolas with Peptide I head group, theconjugation is carried out through the nitrogen of the indole ring orthrough the hydroxyproline residue; for compound II, through the aminogroup; and for compound III, the fatty acid residue will be attachedthrough the carboxylic group, or alternatively through the amino group.In each case, the site of attachment is chosen based on the results ofthe targeting efficacy in vitro studies.

The following provides illustrative examples of compounds useful as NK1Rantagonists.

I {1-[4-(1H-tetrazol-5-yl)butyl]indol-3-yl}carbonyl-Hyp-Nal-N(methyl)-Bz1, (Hyp = (R)-4-hydroxy-L-proline; Nal = 3-L-(β-naphthyl)-alanine) II CP-99,994 III(+)-[2R,3R,4R,8R,9(3′R)-2-{1-[1(3,5bis(tri-fluoromethyl)phenyl]ethyl)oxy}-4(-3carboxy-3-methylpiperidinlyl)-3-phenyl-methyltetra- hydropyran

General Synthetic Procedures

The compounds provided herein can be purchased or prepared from readilyavailable starting materials using the following general methods andprocedures. See, e.g., Synthetic Schemes below. It will be appreciatedthat where typical or preferred process conditions (i.e., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. Optimum reaction conditions may vary with theparticular reactants or solvent used, but such conditions can bedetermined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. The choice of asuitable protecting group for a particular functional group as well assuitable conditions for protection and deprotection are well known inthe art. For example, numerous protecting groups, and their introductionand removal, are described in T. W. Greene and P. G. M. Wuts, ProtectingGroups in Organic Synthesis, Second Edition, Wiley, New York, 1991, andreferences cited therein.

The compounds provided herein may be isolated and purified by knownstandard procedures. Such procedures include (but are not limited to)recrystallization, column chromatography or HPLC. The following schemesare presented with details as to the preparation of representativesubstituted biarylamides that have been listed herein. The compoundsprovided herein may be prepared from known or commercially availablestarting materials and reagents by one skilled in the art of organicsynthesis.

The enantiomerically pure compounds provided herein may be preparedaccording to any techniques known to those of skill in the art. Forinstance, they may be prepared by chiral or asymmetric synthesis from asuitable optically pure precursor or obtained from a racemate by anyconventional technique, for example, by chromatographic resolution usinga chiral column, TLC or by the preparation of diastereoisomers,separation thereof and regeneration of the desired enantiomer. See,e.g., “Enantiomers, Racemates and Resolutions,” by J. Jacques, A.Collet, and S. H. Wilen, (Wiley-Interscience, New York, 1981); S. H.Wilen, A. Collet, and J. Jacques, Tetrahedron, 2725 (1977); E. L. ElielStereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and S. H.Wilen Tables of Resolving Agents and Optical Resolutions 268 (E. L.Eliel ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972,Stereochemistry of Organic Compounds, Ernest L. Eliel, Samuel H. Wilenand Lewis N. Manda (1994 John Wiley & Sons, Inc.), and StereoselectiveSynthesis A Practical Approach, Mihály Nógrádi (1995 VCH Publishers,Inc., NY, N.Y.).

In certain embodiments, an enantiomerically pure compound of formula (1)may be obtained by reaction of the racemate with a suitable opticallyactive acid or base. Suitable acids or bases include those described inBighley et al., 1995, Salt Forms of Drugs and Adsorption, inEncyclopedia of Pharmaceutical Technology, vol. 13, Swarbrick & Boylan,eds., Marcel Dekker, New York; ten Hoeve & H. Wynberg, 1985, Journal ofOrganic Chemistry 50:4508-4514; Dale & Mosher, 1973, J Am. Chem. Soc.95:512; and CRC Handbook of Optical Resolution via Diastereomeric SaltFormation, the contents of which are hereby incorporated by reference intheir entireties.

Enantiomerically pure compounds can also be recovered either from thecrystallized diastereomer or from the mother liquor, depending on thesolubility properties of the particular acid resolving agent employedand the particular acid enantiomer used. The identity and optical purityof the particular compound so recovered can be determined by polarimetryor other analytical methods known in the art. The diastereoisomers canthen be separated, for example, by chromatography or fractionalcrystallization, and the desired enantiomer regenerated by treatmentwith an appropriate base or acid. The other enantiomer may be obtainedfrom the racemate in a similar manner or worked up from the liquors ofthe first separation.

In certain embodiments, enantiomerically pure compound can be separatedfrom racemic compound by chiral chromatography. Various chiral columnsand eluents for use in the separation of the enantiomers are availableand suitable conditions for the separation can be empirically determinedby methods known to one of skill in the art. Exemplary chiral columnsavailable for use in the separation of the enantiomers provided hereininclude, but are not limited to CHIRALCEL® OB, CHIRALCEL® OB-H,CHIRALCEL® OD, CHIRALCEL® OD-H, CHIRALCEL® OF, CHIRALCEL® OG, CHIRALCEL®OJ and CHIRALCEL® OK.

Abbreviations

BBB, blood brain barrier

BCECs, brain capillary endothelial cells

CF, carboxyfluorescein

CHEMS, cholesteryl hemisuccinate

CHOL, cholesterol

Cryo-TEM, Cryo-transmission electron microscope

DAPI, 4′,6-diamidino-2-phenylindole

DDS, drug delivery system

DLS, dynamic light scattering

DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine

DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine

DMPG, 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)

EPR, electron paramagnetic resonance

FACS, fluorescence-activated cell sorting

FCR, fluorescence colorimetric response

GUVs, giant unilamellar vesicles

HPLC, high performance liquid chromatography

IR, infrared

MNPs, Magnetic Nanoparticles

MRI, magnetic resonance imaging

NMR, nuclear magnetic resonance

NPs, nanoparticles

PBS, phosphate buffered saline

PC, phosphatidylcholine

PDA, polydiacetylene.

TMA-DPH, 1-(4 trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene

Example 1 Bolaamphiphile Synthesis

The boloamphiphles or bolaamphiphilic compounds of the invention can besynthesized following the procedures described previously (see below).

Briefly, the carboxylic group of methyl vernolate or vernolic acid wasinteracted with aliphatic diols to obtain bisvernolesters. Then theepoxy group of the vernolate moiety, located on C12 and C13 of thealiphatic chain of vernolic acid, was used to introduce two AChheadgroups on the two vicinal carbons obtained after the opening of theoxirane ring. For GLH-20 (Table 1), the ACh head group was attached tothe vernolate skeleton through the nitrogen atom of the choline moiety.The bolaamphiphile was prepared in a two-stage synthesis: First, openingof the epoxy ring with a haloacetic acid and, second, quaternizationwith the N,N-dimethylamino ethyl acetate. For GLH-19 (Table 1) thatcontains an ACh head group attached to the vernolate skeleton throughthe acetyl group, the bolaamphiphile was prepared in a three-stagesynthesis, including opening of the epoxy ring with glutaric acid, thenesterification of the free carboxylic group with N,N-dimethyl aminoethanol and the final product was obtained by quaternization of the headgroup, using methyl iodide followed by exchange of the iodide ion bychloride using an ion exchange resin.

Each bolaamphiphile was characterized by mass spectrometry, NMR and IRspectroscopy. The purity of the two bolaamphiphiles was >97% asdetermined by HPLC.

Materials. Iron(III) acetylacetonate (Fe(acac)₃), diphenyl ether,1,2-hexadecanediol, oleic acid, oleylamine, and carboxyfluorescein (CF)were purchased from Sigma Aldrich (Rehovot, Israel). Chloroform andethanol were purchased from Bio-Lab Ltd. Jerusalem, Israel.1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DMPG),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol (CHOL),cholesteryl hemisuccinate (CHEMS) were purchased from Avanti Lipids(Alabaster, Ala., USA), The diacetylenic monomer 10,12-tricosadiynoicacid was purchased from Alfa Aesar (Karlsruhe, Germany), and purified bydissolving the powder in chloroform, filtering the resulting solutionthrough a 0.45 μm nylon filter (Whatman Inc., Clifton, N.J., USA), andevaporation of the solvent. 1-(4trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) waspurchased from Molecular Probes Inc. (Eugene, Oreg., USA).

Synthesis of Representative Bolaamphiphilic Compounds

The synthesis bolaamphiphilic compounds of this invention can be carriedout in accordance with the methods described previously (Chemistry andPhysics of Lipids 2008, 153, 85-97; Journal of Liposome Research 2010,20, 147-59; WO2002/055011; WO2003/047499; or WO2010/128504) and usingthe appropriate reagents, starting materials, and purification methodsknown to those skilled in the art. Table 1 lists the representativebolaamphiphilic compounds of the invention.

TABLE 1 Representative Bolaamphiphiles # Structure GLH-3 

GLH-4 

GLH-5 

GLH-6^(a)

GLH-7 

GLH-8 

GLH-9 

GLH-10

GLH-11

 GLH-12^(a)

 GLH-13^(a)

 GLH-13^(a)

GLH-14

GLH-15

GLH-16

GLH-17

GLH-18

GLH-19

GLH-20

GLH-21

GLH-22

GLH-23

GLH-24

GLH-25

GLH-26

GLH-27

GLH-28

GLH-29

GLH-30

GLH-30

GLH-31

GLH-32

GLH-33

GLH-34

GLH-35

GLH-36

GLH-37

GLH-38

 GLH-39^(a)

GLH-40

GLH-41

 GLH-42^(a)

 GLH-43^(a)

GLH-44

GLH-45

GLH-46

GLH-47

GLH-48

 GLH-49^(a)

 GLH-50^(a)

 GLH-51^(a)

 GLH-52^(a)

 GLH-53^(a)

GLH-54

GLH-55

GLH-56

GLH-57

^(a) - an intermediate

Example 2 Bolavesicle Preparation and Characterization

Bolaamphiphiles, cholesterol, and CHEMS (2:1:1 mole ratio) are dissolvedin chloroform or a suitable solvent. 0.5 mg of the biologically activedrug dispersed in chloroform is added to the mix. The solvents areevaporated under vacuum and the resultant thin films are hydrated in 0.2mg/mL CF solution in PBS and probe-sonicated (Vibra-Cell VCX130sonicator, Sonics and Materials Inc., Newtown, Conn., USA) withamplitude 20%, pulse on: 15 sec, pulse off: 10 sec to achieve homogenousvesicle dispersions. Vesicle size and zeta potential were determinedusing a Zetasizer Nano ZS (Malvern Instruments, UK). The amount of thebiologically active drug encapsulated in the vesicles can be determinedby HPLC and/or UV spectroscopy (G Gnanaraian, et al., 2009) afterseparating the non-encapsulated drug, by size exclusion chromatography(on Sephadex-G50).

Spectral Characterization Example 3 Electron Paramagnetic Resonance(EPR)

EPR spectra of biologically active drug embedded bolavesiclesresuspended in PBS can be obtained using a Bruker EMX-220 X-band (υ˜9.4GHz) EPR spectrometer equipped with an Oxford Instruments ESR 900temperature accessories and an Agilent 53150A frequency counter. Spectracan be recorded at room temperature with the non-saturating incidentmicrowave power 20 mW and the 100 KHz magnetic field modulation of 0.2mT amplitude. Processing of EPR spectra, determination of spectralparameters can be done using Bruker WIN-EPR software.

Example 4 Cryogenic Transmission Electron Microscopy (Cryo-TEM)

Specimens studied by cryo-TEM were prepared. Sample solutions (4 μL) aredeposited on a glow discharged, 300 mesh, lacey carbon copper grids (TedPella, Redding, Calif., USA). The excess liquid is blotted and thespecimen was vitrified in a Leica EM GP vitrification system in whichthe temperature and relative humidity are controlled. The samples areexamined at −180° C. using a FEI Tecnai 12 G2 TWIN TEM equipped with aGatan 626 cold stage, and the images are recorded (Gatan model 794charge-coupled device camera) at 120 kV in low-dose mode. FIG. 1 showsTEM micrograph of vesicles from GLH-20 (A) and their size distributiondetermined by DLS (B).

Assays Example 5 Lipid/Polydiacetylene (PDA) Assay

Lipid/polydiacetylene (PDA) vesicles (PDA/DMPC 3:2, mole ratio) areprepared by dissolving the lipid components in chloroform/ethanol anddrying together in vacuo. Vesicles are subsequently prepared in DDW byprobe-sonication of the aqueous mixture at 70° C. for 3 min. The vesiclesamples are then cooled at room temperature for an hour and kept at 4°C. overnight. The vesicles are then polymerized using irradiation at 254nm for 10-20 s, with the resulting emulsions exhibiting an intense blueappearance. PDA fluorescence is measured in 96-well microplates (GreinerBio-One GmbH, Frickenhausen, Germany) on a Fluoroscan Ascentfluorescence plate reader (Thermo Vantaa, Finland). All measurements areperformed at room temperature at 485 nm excitation and 555 nm emissionusing LP filters with normal slits. Acquisition of data is automaticallyperformed every 5 min for 60 min. Samples comprised 30 μL of DMPC/PDAvesicles and 5 μL bolaamphiphilic vesicles assembled with biologicallyactive drug, followed by addition of 30 μL 50 mM Tris-base buffer (pH8.0).

A quantitative value for the increasing of the fluorescence intensitywithin the PDA/PC-labeled vesicles is given by the fluorescencecolorimetric response (% FCR), which is defined as follows²⁷:

% FCR=[(F _(I) −F ₀)/F ₁₀₀]·100  Eq. 1.

Where F_(I) is the fluorescence emission of the lipid/PDA vesicles afteraddition of the tested membrane-active compounds, F₀ is the fluorescenceof the control sample (without addition of the compounds), and F₁₀₀ isthe fluorescence of a sample heated to produce the highest fluorescenceemission of the red PDA phase minus the fluorescence of the controlsample.

Example 6 Cell Culture

b.End3 immortalized mouse brain capillary endothelium cells are kindlyprovided by Prof. Philip Lazarovici (Institute for Drug Research, Schoolof Pharmacy, The Hebrew University of Jerusalem, Israel). The b.End3cells were cultured in DMEM medium supplemented with 10% fetal bovineserum, 2 mM L-Glutamine, 100 IU/mL penicillin and 100 g/mL streptomycin(Biological Industries Ltd., Beit Haemek, Israel). The cells aremaintained in an incubator at 37° C. in a humidified atmosphere with 5%CO₂.

Example 7 Internalization of CF by the Cells In Vitro

b.End3 cells are grown on 24-well plates or on coverslips (for FACS andfluorescence microscopy analysis, respectively). The medium is replacedwith culture medium without serum and CF solution, or testedbolavesicles (equivalent to 0.5 μg/mL CF), or equivalent volume of themedium are added to the cells and incubated for 5 hr at 4° C. or at 37°C. At the end of the incubation, cells are extensively washed withcomplete medium and with PBS, and are either detached from the platesusing trypsin-EDTA solution (Biological Industries Ltd., Beit Haemek,Israel) and analyzed by FACS (FACSCalibur Flow Cytometer, BDBiosciences, USA), or fixed with 2.5% formaldehyde in PBS, washed twicewith PBS, mounted on slides using Mowiol-based mounting solution andanalyzed using a FV1000-IX81 confocal microscope (Olympus, Tokyo, Japan)equipped with 60× objective. All the images are acquired using the sameimaging settings and are not corrected or modified. The FIG. 2 showshead group hydrolysis by AChE (A) of GLH-19 (blue) and GLH-20 (red) andrelease of CF from GLH-19 vesicles (B) and GLH-20 vesicles (C). AChEcauses the release of encapsulated material from GLH-20 vesicles, butnot from GLH-19 vesicles (FIG. 2). The vesicles are capable ofdelivering small molecules, such as carboxyfluorescein (CF), into amouse brain, but the fluorescent dye accumulates only if it is deliveredin vesicles that release their encapsulated CF in presence of AChE,namely, GLH-20 vesicles (FIG. 3A). These results suggest that therelease is due to head group hydrolysis by AChE in the brain.Corroboration for this conclusion also comes from an experiment showingthat when an analgesic peptide is delivered to the brain by the bolavesicles, analgesia (which is caused when the encapsulated peptide isreleased in the brain) was observed only with GLH-20 vesicles, but notby GLH-19 vesicles (FIG. 4A). The vesicles do not break the BBB, butrather penetrate it in their intact form, as indicated by the findingthat analgesia is obtained only when enkephalin is administered whileencapsulated within the vesicles, but not when free enkephalin isadministered together with empty vesicles (FIG. 4B).

The ACh head groups also provide the vesicles with cationic surfaces,which promote penetration through the BBB [Lu et al, 2005] and transportof the encapsulated material into the brain. Toxicity studies showedthat the dose which induced the first toxic signs was 10-20 times higherthan the doses needed to obtain analgesia by encapsulated analgesicpeptides.

The addition of chitosan (CS) surface groups, by employing CS-vemolateconjugates, increased BBB permeability of the vesicles (FIG. 4B),probably by increasing transcytosis [Newton, 2006]. However, the CSgroups, when added to the vesicles by employing fatty acid-CS conjugate(in this case, vernolic acid), are not stable in circulation as surfacegroups because of the low energy barrier for lipid exchange of suchconjugates. The inventors propose to make stabilized CS surface groupsby using bolas that the inventors will synthesize withcovalently-attached CS head groups [see, Experimental Design andMethods, below].

In addition to the peptide leu-enkephalin, and the small molecules: CF,uranyl acetate, kyotorphin and sucrose, the inventors have alsosuccessfully encapsulated in these vesicles the proteins albumin andtrypsinogen and the polysaccharide Dextran-FITC (MW 9000). Albumin-FITC,encapsulated, was delivered successfully to the brain (FIG. 5B), whileun-encapsulated albumin-FITC showed little, if any, brain accumulation(FIG. 5A), indicating that the vesicle transported the protein into thebrain through the BBB. These results strongly suggest that the vesiclescan be made to encapsulate other molecules, such as anti-retroviraldrugs, and deliver them into the brain without harming the BBB.

Example 8 Statistical Analysis

The data are presented as mean and standard deviations (SD) or standarderrors of mean (SEM). Statistical differences between the control andthe studied formulations are analyzed using ANOVA followed by Dunnettpost-test using InStat 3.0 software (GraphPad Software Inc., La Jolla,Calif., USA). P values of less than 0.05 are defined as statisticallysignificant.

Example 9 Encapsulation of CPT-11

A) Optimization of vesicle formation: Vesicles are prepared by filmhydration, followed by sonication. Each of the vesicle formulations canbe examined for vesicle size (by dynamic light scattering), morphology(by cryo-transmission electron microscopy), zeta potential (by ZetaPotential Analyzer) and stability (by fluorescence measurements ofencapsulated CF at various times after vesicle preparation). Stabilityof vesicles can be determined in presence and absence of ChE, with andwithout an inhibitor of the enzyme (e.g., pyridostigmine).

B) Encapsulation of CPT-11: To successfully encapsulate CPT-11 (MW586.67, water solubility of 25 mg/ml with bis-piperidine moiety, whichforms an ammonium salt in acid) within the vesicles, the active loadingapproach can be used. CPT-11 can be encapsulated in its active lactonicform, and not in the inactive carboxylate form. The loading conditionsbased on conditions developed for liposomal formulations using a pHgradient between the liposome core can be used and the bathing medium,whereas the internal volume can be acidic compared to the externalsolution.

For encapsulations, vesicles can be formed in acidic buffers, such ascitrates. The vesicles can be purified on a GPC column to separateencapsulated CPT-11 from non-encapsulated material. Percentencapsulation can be determined by UV absorption of the CTP-11'saromatic groups after lysis with a detergent. To maximize CPT-11 loadingand minimize leakage, the composition of the vesicle's membrane can beoptimized by varying both the ratio between bolaamphiphiles in thevesicle formulation and the proportion of different additives used inthe vesicle formulation, such as cholesterol hemisuccinate and neutralcholesterol; or drug-loading with respect to the relative concentrationof CPT-11 to vesicles, the temperature during loading, internal buffercomposition and the pH gradient across the vesicle's membrane.

The entrapped CPT-11 may be stabilized by adding, to the vesicle core,agents that help to prevent leakage, such as dextran sulfate28, coppersulfate and other transition metal salts29, and polymeric or highlycharged nonpolymeric polyanionic trapping agents.

Example 10 Determination of Encapsulated CPT-11 Activity

To ensure that the encapsulation process did not reduce the cytotoxicactivity of CPT-11, the encapsulated CPT-11 can be released from thevesicles by ChE treatment, and the released CPT-11 can be collected fromthe supernatant following centrifugation. The IC₅₀ of the releasedCPT-11 can be determined by using U87 glioblastoma cell line and by astandard viability assay (e.g., MTT) in comparison to that of standardCPT-11.

Example 11 Synthesis of Bolaamphiphiles from Jojoba Oil

This example describes the synthesis of three new, illustrative,bolaamphiphiles from jojoba oil, which are designated GLH-58, GLH-59,and GLH-60, and are depicted below.

GLH-58

GLH-59

GLH-60

We have described novel bolaamphiphiles with acetylcholine (ACh) headgroups and shown that these bolaamphiphiles interact with smallinterference RNA molecules (siRNA) and form particles that areinternalized by cells and silence genes following their internalizationboth in vitro and in vivo. These studies indicated that the ACh headgroups play a major role in the interactions between the siRNA and thebolaamphiphile and additions of head groups may increase the amount ofthe siRNA that binds the bolaamphiphile. The present disclosuredescribes the synthesis of a bolaamphiphile with more than two ACh headgroups and the investigation thereof with respect to their interactionswith siRNA.

The bolaamphiphiles described in previous sections were synthesized fromfatty acids derived from triglyceride vegetable oils (i.e. vernolic andoleic acids). This is a multistage synthesis, since when fatty acidsderived from triglyceride oils are used as the starting material for thesynthesis of bolaamphiphiles, the skeleton of the bolaamphiphile has tobe synthesized first and only then, the ACh head groups are attached tothe bolaamphiphilic skeleton.

In order to simplify the synthesis of boloaamphiphiles with ACh headgroups, particularly bolaamphiphiles with more than two ACh head groups,we used jojoba oil as the starting material

(Z,Z)—CH₃(CH₂)₇CH═CH(CH₂)_(m)COO(CH₂)_(n)CH═CH(CH₂)₇CH₃   Jojoba oil

-   -   m=7, 9, 11, 13    -   n=8, 10, 12, 14

In contrast to the triglyceride vegetable oils, jojoba oil is a liquidwax with a 40-42 carbon atom chain composed mainly of straight chainmonoesters of C₂₀ and C₂₂ monounsaturated acids and alcohols. Jojoba oilconstitutes a unique starting material for the synthesis ofbolaamphiphiles as its chemical structure may provide a hydrophobicskeleton of 40-44 carbon atoms and the ACh head groups can be bounddirectly to the jojoba oil, which is used as the bolaamphiphilicskeleton.

The two double bonds on either side of the jojoba's aliphatic chain areused to attach the head groups. The ACh head groups can be attached tothe jojoba skeleton in two different ways: (a) direct addition ofhaloacetic acid to the double bond followed by quaternization of thehead group, or (b) epoxidation of the double bonds and opening the epoxygroup; e.g. esterification of the hydroxyl groups formed with ahaloacetic acid followed by quaternization of the tertiary amine to givea bolaamphiphilic compound. Two examples are provided in the followingstructures:

The chemical structure of bolaamphiphilic compounds with ACh head groupsthat were synthesized from jojoba oil include the above structures,where compound (a) is designated as GLH-58, a bolaamphiphile with twoACh head groups, and compound (b) is designated GLH-60, a bolaamphiphilewith four ACh head groups.

Example 12 Synthesis of the Bolaamphiphilic Compound, GLH-58

In this Example, the bolaamphiphilic compound, GLH-58 was synthesizedthrough a direct addition of a halo acetic acid to the double bonds ofjojoba oil. A first step involved synthesis of the dichloroacetatederivative of jojoba oil. In one embodiment, the method described byCarey [Carey F. A., Sundberg R. J. Advanced Organic Chemistry fifthedition, Part A: Structure and Mechanisms. Chapter 5. Polar Addition andElimination Reactions (2008): 473-477] for a direct addition ofchloroacetic acid to double bonds was employed. However, the addition ofchloroacetic acid to jojoba oil under these conditions (without using acatalyst) did not result in the formation of a product. Therefore, thereaction has been performed under acidic conditions, in the presence ofa concentrated H₂SO₄, or in the presence of a cation exchange resin[Patwardhan A. A, Sharma M. M., Esterification of Carboxylic Acids withOlefins using Cation Exchange Resins as Catalysts. Reactive Polymers, 13(1990): 161-176, and Chakrabarti A., Sharma M. Esterification of AceticAcid with Styrene: Ion Exchange Resins as Catalysts. Reactive Polymers,16 (1991/1992): 51-59]. We found that Jojoba oil (compound 1 in Scheme1, below) reacted with a threefold excess of chloroacetic acid (compound2 in Scheme 1) at 90° C. in the presence of the catalyst Amberlyst 15,which was dried by toluene azeotropic distillation.

The progress of the reaction was followed by monitoring the products onTLC and HPLC. The appearance of two new products in addition to thestarting material was observed after about two hours. The two productswere isolated by a flash column chromatography and identified as jojobamonochloroacetate (compound 3 in Scheme 1) and jojoba dichloroacetate(compound 4 in Scheme 1).

The FT-IR spectrum of the jojoba monochloroacetate 3 showed the peakscharacteristic of a double bond at 3006 cm-1, of a carbonylic estergroup at 1737 cm-1, and a new chloroacetate ester group at 1759, 1289and 1254 cm-1. By comparison, the FT-IR spectrum of jojobadichloroacetate 4 showed the disappearance of the absorption bandscharacteristic to the double bond and the appearance of the newabsorption band for the new chloroacetate ester groups, very similar tothose of the jojoba monochloroacetate 3. The ratio of the peak area ofthe chloroacetate (1758 cm-1)/to the peak area of the original estergroup of jojoba (1735 cm-1) in FT-IR was found to be equal to 0.3 forthe monochloroacetate 3 and 0.6 for the dichloroacetate 4.

The NMR spectrum of jojoba dichloroacetate 4 showed the disappearance ofthe double bonds at 5.2 ppm. The new chemical shifts characteristic ofthe CH moiety of the new ester group: CH—O—CO—CH₂—Cl appeared as aquintet at 4.94 ppm in ¹H-NMR and at 75.9 ppm in ¹³C-NMR; thechloromethylene group CH—O—CO—CH₂—Cl as singlet at 4.23 ppm and at 41.00ppm, correspondingly, and the new carbonyl group: C═O—CH₂—Cl at 166.88ppm (FIG. 7).

The HPLC chromatogram of the products showed five main peaks, indicatingon 5 components of the jojoba dichloroacetate derivatives. The differentcomponents of the reaction mixture were identified by MALDI-MS (FIG.8(a)) as the jojoba dichloroacetate derivatives 4 (C₄₈H₉₀Cl₂O₆,C₄₆H₈₆Cl₂O₆, C₄₄H₈₂Cl₂O₆, C₄₂H₇₈Cl₂O₆, C₄₀H₇₄Cl₂O₆). The ratio of theisotopes in the main compound of this mixture (C₄₆H₈₆Cl₂O₆ ⁺Na) wasconsistent with the calculated value (FIG. 8(b)). The abundance ofdichloroacetate derivatives corresponds to the abundance of originaljojoba oil molecules.

Synthesis of bolaamphiphile GLH-58: In the last stage of the synthesis,the jojoba oil dichloroacetate 4 was used as the alkylating agent forthe quaternization of the tertiary amine N,N′-dimethylaminoethyl acetate(compound 5 in Scheme 2). The Jojoba dichloroacetate 4 was reacted withan excess of the amine 5 at 60° C. for 5 h to obtain the bolaamphiphileGLH-58 that contains two ACh head groups as depicted in Scheme 2:

TLC of the reaction mixture showed a new compound already after 2h, andafter 5h all the dichloroacetate derivatives 4 were consumed. Thereaction mixture was cooled to room temperature; hexane was added toremove the excess of the amine 5. The hexane extraction process wasrepeated several times. The lower phase, containing the crude product,was collected and the solvent was removed under reduced pressure andfurther purified by flash chromatography using acetonitrile:water (10:1)as the eluent. The purity of the GLH-58 was 98.4%, as determined byargentometric titration and its appearance was viscous liquid.

In the FT-IR spectra, the absorption bands, characteristic of thechloroacetate ester, at 1757, 1290, 1257, and 1184 cm-1 and of the C—Clbond at 784 cm¹ disappeared, and new absorption bands appeared at 3383cm⁻¹, 3017 cm-1 and are attributed to the C—H stretch of thenitrogen-bound methyl groups. The chemical shifts characteristic of thechloromethylene group (—CH₂—Cl) of intermediate 4 at 4.23 ppm and 41.00ppm in the ¹H- and ¹³C-NMR, respectively, disappeared and new signals ofthe quaternary ammonium group appeared (FIG. 9). The chemical shifts ofthe methyl groups of the quaternary nitrogen (CH₃)₂N⁺ appeared at 3.68and 52.78 ppm in the ¹H-NMR and ¹³C-NMR spectra, respectively, and forCH—O—C═O at 4.90 ppm and 78.26 ppm, respectively. The chemical shiftsfor the methyl of the ACh group CH₃—C(O)—O appeared at 2.10 and 20.88ppm, and for the carbonyl carbon CH₃—C(O)—O, at 169.88 ppm (FIG. 9).

The MS spectrum of GLH-58 (FIG. 10) showed the presence of three mainpeaks for the quaternary bolaamphiphile [M-2Cl/2]⁺: 486.4, 506.2 and514.5 for C₅₈H₁₁₂O₁₈N₂Cl₂, C₅₉H₁₁₄O₁₀N₂Cl₂ and C₆₀H₁₁₆O₁₀N₂Cl₂,respectively. These results are consistent with the theoreticalmolecular mass of the bolaamphiphile with the two ACh head groupsderived from the corresponding jojoba dichloroacetate main molecules.

Example 13 Synthesis of bolaamphiphile GLH-60 through epoxidation ofjojoba oil

GLH-60, a bolaamphiphilic compound with four ACh head groups, wassynthesized as depicted in Scheme 3, below, using jojoba diepoxide 7 asthe starting material.

Synthesis of jojoba diepoxide 7: The epoxidation of jojoba oil wascarried out using an excess of m-chloroperbenzoic acid (m-CPBA)-compound6 in Scheme 3 [Lynch B. M. and Pausacker K. H., J. Chem. Soc., (1955):1525; Kim C. C., Traylor T. G, and Perrin. C. L. MCPBA Epoxidation ofAlkenes: Reinvestigation of Correlation between Rate and IonizationPotential. J. Am. Chem. Soc. 120 (1998): 9513-9516; Eugeniuzs M.,Smagowicz A., Lewandowski G. Optimization of the Epoxidation of Rapeseedoil with Peracetic Acid. Organic Process Research & Development (2010):1094-1101]. The reaction was performed in CHCl₃ at 5-10° C. andmonitored by thin layer chromatography (TLC). After two hours the totaldisappearance of the double bond, characteristic of jojoba oil, and theappearance of a new polar compound was observed. The jojoba diepoxide(compound 7 in Scheme 3) was obtained in a 74.6% yield and 84.5% purityas determined by potentiometric titration.

The FT-IR of jojoba diepoxide 7 showed the typical epoxy groupabsorption bands at 820 and 842 cm⁻¹ and the disappearance of theabsorption peak at 3004 cm⁻¹ the C—H stretching in the double bond.

In the NMR spectrum, the peak of the double bond of jojoba oildisappeared and a new signal, characteristic of the epoxy group protons,appeared at 2.77 ppm and 57.29 ppm in the ¹H- and ¹³C-NMR spectra,respectively.

Synthesis of tetrahydroxy jojoba oil: The hydrolysis of epoxides is pHdependent and can occur through acid, neutral or base promotedprocesses, but the acid and neutral processes dominate overenvironmentally significant pH ranges [Rogers E. Harry-O'kurua,Abdellatif Mohamedb, Thomas P. Abbott. Synthesis and Characterization ofTetrahydroxy Jojoba Wax and Ferulates of Jojoba Oil. Science 22 (2005):125-133]. The hydrolysis of jojoba diepoxide by the opening of the epoxygroups to form a diol on each side of the ester (scheme 3) was carriedout in the presence of concd. H₂SO₄. After washing and precipitation ofthe product with petroleum ether, the tetrahydroxy jojoba oil 8 wasobtained as a white powder in 81% yield.

The FT-IR spectrum of intermediate 8, the tetrahydroxy jojoba oil,showed absorption bands characteristic of the —OH at 3310 cm⁻¹ and forC—O—C at 1183 cm⁻¹. In ¹H-NMR and ¹³C-NMR was observed the CH—OH groupat 3.37 and 74.40 ppm respectively.

Synthesis of tetrachloroacetate of jojoba oil 10: The esterification oftetrahydroxy jojoba oil (compound 8 in Scheme 3) was performed by usingan excess of chloroacetyl chloride (compound 9 in Scheme 3), inchloroform as the solvent at 0° C. in the presence of pyridine (scheme3). The tetrachloroacetate of jojoba oil (compound 10 in Scheme 3) wasseparated from the reaction mixture by flash column chromatography,using chloroform as the eluting solvent, and appeared as a yellowsemi-solid which was obtained in 59% yield.

FT-IR spectra of compound 10 showed that the absorption bands,characteristic of the hydroxyl groups, disappeared and new absorptionbands, characteristic of the chloroacetate group, appeared at 1762 (C═O)and 1286 cm⁻¹ (C—O).

The NMR analysis showed new chemical shifts, characteristics of themethane proton CH—O—CO—CH₂—Cl, as multiplet at 5.02 ppm and at 75.11ppm, in 1H- and 13C-NMR, respectively. The chloromethylene groupCH—O—CO—CH₂—Cl appeared as a singlet at 4.19 ppm and at 40.68 ppm in ¹H-and ¹³C-NMR, respectively and the new carbonyl group C═O—CH₂—Cl at166.79 ppm (FIG. 11).

The MALDI-MS of compound 10, C₅₀H₈₈O₁₀Cl₄ and C₄₈H₈₄O₁₀Cl₄ (FIG. 12) isconsistent with the theoretical molecular mass of tetrachloroacetate ofjojoba oil 10 derived from the esters with 42 carbons and 40 carbons.The isotope abundance pattern for each molecular weight corresponds fora molecule containing four chlorine atoms.

Synthesis of bolaamphiphile GLH-60: In the last stage of the synthesisthe tetrachloroacetate intermediate 10 was reacted with a small excessof N,N′-dimethylaminoethyl acetate 5 at 60° C. for 6 h to obtain thebolaamphiphile GLH-60 with four ACh head groups (Scheme 3). Thenon-reacted N,N′-dimethylaminoethyl acetate was separated from the crudeproduct by adding hexane followed by decantation as was described above.The bolaamphiphilic compound, GLH-60, was obtained as a viscous liquidwith a purity of 96%, as determined by argentometric titration.

The MALDI MS of GLH-60: m/z [M-4Cl/4]⁺:336.4, 342.4, 350.4 and 357.4 forC₇₂H₁₃₆O₁₈N₄Cl₄, C₇₄H₁₄₀O₁₈N₄Cl₄, C₇₆H₁₄₄O₁₈N₄Cl₄, C₇₈H₁₄₈O₁₈N₄Cl₄ wasconsistent with the theoretical molecular mass of a bolaamphiphile withthe four ACh head groups derived from the corresponding esters of jojobaoil (FIG. 12). Jojoba Tetrachloroacetate is compound 10 in Scheme 3.

As described here a novel formulations of bolavesicles can be producedthrough co-assembly of biologically active drugs withbolaamphiphile/lipid unilamellar vesicles. The formulations can beexamined for their chemical and biophysical properties.

The incorporation of biologically active drug within the bolavesicles isshown to significantly modulate interactions with membrane bilayers inmodel systems. This observation is important, suggesting thatbiologically active drugs encapsulated in bolavesicles might beexcellent candidates for targeting and transport of different molecularcargoes into the brain.

From the foregoing description, various modifications and changes in thecompositions and methods provided herein will occur to those skilled inthe art. All such modifications coming within the scope of the appendedclaims are intended to be included therein.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

At least some of the chemical names of compounds of the invention asgiven and set forth in this application, may have been generated on anautomated basis by use of a commercially available chemical namingsoftware program, and have not been independently verified.Representative programs performing this function include the Lexichemnaming tool sold by Open Eye Software, Inc. and the Autonom Softwaretool sold by MDL, Inc. In the instance where the indicated chemical nameand the depicted structure differ, the depicted structure will control.

Chemical structures shown herein were prepared using ISIS®/DRAW. Anyopen valency appearing on a carbon, oxygen or nitrogen atom in thestructures herein indicates the presence of a hydrogen atom. Where achiral center exists in a structure but no specific stereochemistry isshown for the chiral center, both enantiomers associated with the chiralstructure are encompassed by the structure.

REFERENCES

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1.-88. (canceled)
 89. A pharmaceutical composition or a formulationcomprising a bolaamphiphile complex, a sub-micron sized vesicle, ornano-sized vesicle; wherein the bolaamphiphile complex or nano-sizedvesicles comprises one or more bolaamphiphilic compounds and abiologically active compound, wherein the bolaamphiphilic compound is acompound according to formula I:HG²-L¹-HG¹   I or a pharmaceutically acceptable salt, solvate, hydrate,prodrug, stereoisomer, tautomer, isotopic variant, or N-oxide thereof,or a combination thereof; wherein: each HG¹ and HG² is independently ahydrophilic head group; and L¹ is alkylene, alkenyl, heteroalkylene, orheteroalkenyl linker; unsubstituted or substituted with C₁-C₂₀ alkyl,hydroxyl, or oxo.
 90. The pharmaceutical composition according to claim89, wherein L¹ is heteroalkylene, or heteroalkenyl linker comprising C,N, and O atoms; unsubstituted or substituted with C₁-C₂₀ alkyl,hydroxyl, or oxo.
 91. The pharmaceutical composition according to claim89, wherein the bolaamphiphilic compound is a compound according toformula II, III, IV, V, or VI:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof; wherein: each HG¹ and HG² is independently ahydrophilic head group; each Z¹ and Z² is independently —C(R³)₂—,—N(R³)— or —O—; each R^(1a), R^(1b), R³, and R⁴ is independently H orC₁-C₈ alkyl; each R^(2a) and R^(2b) is independently H, C₁-C₈ alkyl, OH,alkoxy, or O-HG¹ or O-HG²; each n8, n9, n11, and n12 is independently aninteger from 1-20; n10 is an integer from 2-20; and each dotted bond isindependently a single or a double bond.
 92. The pharmaceuticalcomposition according to claim 91, wherein the bolaamphiphilic compoundis a compound according to formula II, III, IV, V, or VI; and each n8and n12 is independently 1, 2, 3, or
 4. 93. The pharmaceuticalcomposition according to claim 91, wherein the bolaamphiphilic compoundis a compound according to formula II, III, IV, V, or VI; and eachR^(2a) and R^(2b) is independently H, OH, alkoxy, or O-HG¹ or O-HG². 94.The pharmaceutical composition according to claim 91, wherein thebolaamphiphilic compound is a compound according to formula II, III, IV,V, or VI; and each R^(1a) and R^(1b) is independently H, Me, Et, n-Pr,i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl, n-heptyl, orn-octyl.
 95. The pharmaceutical composition according to claim 91,wherein the bolaamphiphilic compound is a compound according to formulaII, III, IV, or V; n10 is an integer from 2-16.
 96. The pharmaceuticalcomposition according to claim 91, wherein the bolaamphiphilic compoundis a compound according to formula II, III, IV, V, or VI; and each Z¹and Z² is —O—.
 97. The pharmaceutical composition according to claim 89,wherein the bolaamphiphilic compound is a compound according to formulaII, III, IV, V, or VI; and each HG¹ and HG² is independently selectedfrom:

wherein: X is —NR^(5a)R^(5b), or —N⁺R^(5a)R^(5b)R^(5c); each R^(5a), andR^(5b) is independently H or substituted or unsubstituted C₁-C₂₀ alkylor R^(5a) and R^(5b) may join together to form an N containingsubstituted or unsubstituted heteroaryl, or substituted or unsubstitutedheterocycle; each R^(5C) is independently substituted or unsubstitutedC₁-C₂₀ alkyl; each R⁸ is independently H, substituted or unsubstitutedC₁-C₂₀ alkyl, alkoxy, or carboxy; m1 is 0 or 1; and each n13, n14, andn15 is independently an integer from 1-20.
 98. The pharmaceuticalcomposition according to claim 89, wherein the bolaamphiphilic compoundis a compound according to formula VIIIa, VIIIb, VIIIc, or VIIId:

or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,stereoisomer, tautomer, isotopic variant, or N-oxide thereof, or acombination thereof; wherein: each X is —NR^(5a)R^(5b), or—N⁺R^(5a)R^(5b)R^(5c); each R^(5a), and R^(5b) is independently H orsubstituted or unsubstituted C₁-C₂₀ alkyl or R^(5a) and R^(5b) may jointogether to form an N containing substituted or unsubstitutedheteroaryl, or substituted or unsubstituted heterocycle; each R^(5C) isindependently substituted or unsubstituted C₁-C₂₀ alkyl; n10 is aninteger from 2-20; and each dotted bond is independently a single or adouble bond.
 99. The pharmaceutical composition according to claim 97,wherein each R^(5a), R^(5b), and R^(5c) is independently substituted orunsubstituted C₁-C₂₀ alkyl.
 100. The pharmaceutical compositionaccording to claim 97, wherein X is —N(Me)-CH₂CH₂—OAc or—N⁺(Me)₂-CH₂CH₂—OAc.
 101. The pharmaceutical composition according toclaim 97, wherein X is a headgroup comprising NK1R antagonist.
 102. Thepharmaceutical composition according to claim 97, wherein X is aheadgroup comprising NK1R antagonist, and the NK1R antagonist is I, II,or III:

I {1-[4-(1H-tetrazol-5-yl)butyl]indol-3-yl}carbonyl-Hyp-Nal-N(methyl)-Bz1, (Hyp = (R)-4-hydroxy-L-proline; Nal = 3-L-(β-naphthyl)-alanine) II CP-99,994 III(+)-[2R,3R,4R,8R,9(3′R)-2-{1-[1(3,5bis(tri-fluoromethyl)phenyl]ethyl)oxy}-4(-3carboxy-3-methylpiperidinlyl)-3-phenyl-methyltetra- hydropyran


103. The pharmaceutical composition according to claim 89, wherein thebolaamphiphilic compound is a pharmaceutically acceptable salt.
 104. Thepharmaceutical composition according to claim 89, wherein thebolaamphiphilic compound is in a form of a quaternary salt.
 105. Thepharmaceutical composition of claim 89, wherein the biologically activecompound is a drug active against brain tumor.
 106. The pharmaceuticalcomposition of claim 89, wherein the biologically active compound isCPT-11, or BCNU (Carmustine).